Process for preparing glycopeptides having asparagine-linked oligosaccharides, and the glycopeptides

ABSTRACT

Glycopeptide having at least one asparagine-linked oligosaccharide at a desired position of the peptide chain obtained by:
         (1) esterifying hydroxyl of a resin and carboxyl of ah amino acid having amino group nitrogen protected with a fat-soluble protective group (AGFPG),   (2) removing the protective group to form a free amino group,   (3) amidating the free amino group and carboxyl of an amino acid having AGFPG,   (4) removing the protective group,   (5) repeating the steps (3) and (4),   (6) amidating the free amino group and carboxyl of the asparagine portion of an asparagine-linked oligosaccharide having AGFPG,   (7) removing the protective group,   (8) amidating the free amino group and carboxyl of an amino acid having AGFPG,   (9) repeating steps (7) and (8),   (10) removing the protective group, and   (11) cutting off the resin with an acid;   glycopeptide obtained by transferring sialic acid or a derivative thereof to the above glycopeptide.

This application is a division of application Ser. No. 10/519,983, filedJan. 4, 2005, which is a 371 of PCT/JP2003/008551 filed Jul. 4, 2003,which claims priority based on Japanese patent application Nos.2002-196821 and 2002-349166 filed July 5 and Nov. 29, 2002,respectively, and which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a process for preparing glycopeptideshaving asparagine-linked oligosaccharides, and to the glycopeptideswhich is obtainable by the process.

BACKGROUND ART

In recent years, molecules of oligosaccharides have attracted attentionas third chain life molecules following nucleic acids (DNA) andproteins. The human body is a huge cell society comprising about 60trillion cells, and the surfaces of all the cells are covered witholigosaccharide molecules. For example, ABO blood groups are determinedaccording to the difference of oligosaccharides over the surfaces ofcells.

Oligosaccharides function in connection with the recognition of cellsand interaction of cells and are key substances for the establishment ofthe cell society. Disturbances in the cell society lead, for example, tocancers, chronic diseases, infectious diseases and aging.

For example, it is known that when cells develop cancer, changes occurin the structure of oligosaccharides. It is also known that Vibriocholerae, influenza virus, etc. ingress into cells and cause infectionby recognizing and attaching to a specific oligosaccharide.

Clarification of oligosaccharide functions leads to development ofpharmaceuticals and foods based on novel principles, contributing to theprevention and therapy of diseases, and a wide variety of applicationsare expected of oligosaccharides.

Oligosaccharides are much more complex than nucleic acids or proteins instructure because of the diversity of arrangements of simple sugars,modes or sites of linkages, lengths of chains, modes of branches andoverall structures of higher order. Accordingly, biological informationderived from the structures thereof is more diversified than is the casewith nucleic acids and proteins. Although the importance of research onoligosaccharides has been recognized, the complexity and variety ofstructures thereof have delayed progress in the research onoligosaccharides unlike the studies on nucleic acids and proteins.

Many of proteins present on the surfaces of cell membranes or in serumhave oligosaccharides attached thereto as described above. The moleculeswherein oligosaccharides are combined covalently with proteins aretermed glycoproteins, which can be divided into two groups according tothe difference in the mode of linkage between the oligosaccharide andthe protein. Oligosaccharides of one type are asparagine-linkedoligosaccharides (N-glycoside linkage type) wherein an amino group ofthe side chain of asparagine (Asn) is linked with the oligosaccharide.Oligosaccharides of the other type are mucin-linked oligosaccharides(O-glycoside linkage type) wherein the oligosaccharide is linked withthe alcohol of serine (Ser) or threonine (Thr). All theasparagine-linked oligosaccharides have a basic skeleton comprising fivesugar residues, and are divided into subgroups of high-mannose type,composite type and mixture type, according to the kind of thenonreducing terminal sugar residue of the oligosaccharide linked. On theother hand, the mucin-liked oligosaccharides are divided into fourgroups according to the difference of the basic skeleton.

The process for preparing peptides which is presently in wide use is thesolid-phase synthesis process developed by R. B. Merrifield in 1963. Thesolid-phase synthesis process is such that amino acids are linked to asolid phase called a resin to provide a lengthened peptide chain. Whencompletely lengthened, the peptide chain is cut off from the solid phaseto obtain the desired product. As an application of this process, aglycopeptide chain can be prepared by incorporating an amino acid havingan oligosaccharide linked thereto into the peptide chain to belengthened.

Accordingly, glycopeptide chains are widely prepared by using aminoacid-linked oligosaccharides wherein an oligosaccharide is linked withAsn or Ser(Thr) for the preparation of peptides. However, there are onlya few examples of chemically preparing peptide chains having a greatsugar chain despite of technical progress in chemical synthesis.

One of the problems to be encountered is insufficient absolute amountsof oligosaccharides to be linked with the asparagine residue. Methods ofobtaining oligosaccharides include isolation of oligosaccharides onlyfrom glycoproteins which are present in the living body. However,hydrazine for use in cutting off oligosaccharides from glycoproteins ishazardous, presenting difficulty in preparing large quantities ofoligosaccharides. Further there are in the living body manyoligosaccharides which closely resemble in structure, and it isdifficult to obtain a single oligosaccharide only. Further sincedecomposition of hydrazine releases the oligosaccharide from theasparagine residue, there arises a need to link the releasedoligosaccharide with the asparagine residue again, hence an increasednumber of steps needed.

In chemically synthesizing oligosaccharides, there are examples ofpreparing oligosaccharides wherein about 10 sugar residues are linked,whereas many of these cases are such that the desired oligosaccharidecan be prepared in an amount of only several milligrams during one year.For this reason, difficulties are encountered in chemically preparingoligosaccharides.

The second of the problems is involved in the treatment conducted withuse of TFA (trifluoroacetic acid) for cutting off the peptide chain fromthe solid phase. For example, sialic acid present at the nonreducingterminals of oligosaccharides is readily hydrolyzed under an acidcondition, so that there is the possibility that the TFA treatment willcut off sialic acid from the glycopeptide prepared. Accordingly, thereis almost no case wherein oligosaccharides having sialic acid are usedfor solid-phase synthesis. To solve this problem, a process has beenreported wherein sialic acid is transferred to an oligosaccharide withsialic acid transferase after peptide synthesis. Although useful forintroducing sialic acid, this process still has the problem thatdifficulty is encountered in preparing glycopeptides in large quantitiesbecause the transferase is expensive.

As will be described below, however, the present invention has made itpossible to artificially prepare glycopeptides in large amounts.Accordingly, it becomes possible to industrially introduce sialic acidor derivatives thereof into oligosaccharides using the sialic acidtransferase.

Although there are naturally occurring oligosaccharides which havesialic acid linked thereto, oligosaccharides having sialic acidderivatives linked thereto are naturally unavailable. Thus, it isthrough the use of the sialic acid transferase that sialic acidderivatives can be introduced into oligosaccharides in any way.

An object of the present invention is to provide a process capable ofartificially and easily preparing a large amount of a glycopeptidehaving at least one asparagine-linked oligosaccharide or mucin-linkedoligosaccharide at a desired position of the peptide chain thereof.

Another object of the present invention is to provide a process foreasily preparing a sialylglycopeptide which comprises anasparagine-linked oligosaccharide having sialic acid and wherein thesialic acid is not cut off from the glycopeptide by an acid treatment.

Another object of the present invention is to provide a process forartificially and easily preparing a large quantity of a glycopeptidehaving at least one of various novel asparagine-linked oligosaccharidesat a desired position of the peptide chain thereof, with sugar residuesremoved therefrom as desired.

Another object of the present invention is to provide a process forpreparing a glycopeptide having sialic acid or a derivative thereofintroduced into the peptide with use of a sialic acid transferase.

Still another object of the invention is to provide glycopeptides whichis obtainable by the above processes for preparing glycopeptides.

DISCLOSURE OF THE INVENTION

The present invention provides a process for preparing a glycopeptidehaving at least one asparagine-linked oligosaccharide at a desiredposition of the peptide chain thereof, the process comprising:

(1) esterifying a hydroxyl group of a resin having the hydroxyl groupand a carboxyl group of an amino acid having amino group nitrogenprotected with a fat-soluble protective group,

(2) removing the fat-soluble protective group to form a free aminogroup,

(3) amidating the free amino group and a carboxyl group of an amino acidhaving amino group nitrogen protected with a fat-soluble protectivegroup,

(4) removing the fat-soluble protective group to form a free aminogroup,

(5) repeating the steps (3) and (4) at least once,

(6) amidating the free amino group and a carboxyl group of theasparagine portion of an asparagine-linked oligosaccharide having aminogroup nitrogen protected with a fat-soluble protective group,

(7) removing the fat-soluble protective group to form a free aminogroup,

(8) amidating the free amino group and a carboxyl group of an amino acidhaving amino group nitrogen protected with a fat-soluble protectivegroup,

(9) repeating the steps (7) and (8) at least once,

(10) removing the fat-soluble protective group to form a free aminogroup, and

(11) cutting off the resin with an acid.

The present invention provides a process for preparing a glycopeptidehaving at least two asparagine-linked oligosaccharides at a desiredposition of the peptide chain thereof which comprises the processwherein the steps (6) of amidating the free amino group and a carboxylgroup of the asparagine portion of an asparagine-linked oligosaccharidehaving amino group nitrogen protected with a fat-soluble protectivegroup, and (7) of removing the fat-soluble protective group to form afree amino group are additionally performed suitably.

The present invention provides a process for preparing a glycopeptidehaving at least one asparagine-linked oligosaccharide at a desiredposition of the peptide chain thereof wherein the steps (6) of amidatingthe free amino group and the carboxyl group of the asparagine portion ofan asparagine-linked oligosaccharide having amino group nitrogenprotected with a fat-soluble protective group, and (7) of removing thefat-soluble protective group to form a free amino group are performed asfinal steps.

The present invention provides a process for preparing a glycopeptidewherein the step (1) of esterifying a hydroxyl group of a resin havingthe hydroxyl group and a carboxyl group of an amino acid having aminogroup nitrogen protected with a fat-soluble protective group isperformed in place of the step (6) or in addition to the step (6).

The present invention provides a process for preparing a glycopeptidewherein the asparagine-linked oligosaccharide of the step (6) has atleast 6 sugar residues.

The present invention provides a process for preparing a glycopeptidewherein the asparagine-linked oligosaccharide of the step (6) has 9 to11 sugar residues.

The present invention provides a process for preparing a glycopeptidewherein the asparagine-linked oligosaccharide of the step (6) has atleast 6 sugar residues, and has a bifurcated oligosaccharide attachedthereto.

The present invention provides a process for preparing a glycopeptidewherein the asparagine-linked oligosaccharide in (6) is anasparagine-linked disialooligosaccharide or an asparagine-linkedmonosialooligosaccharide in which the carboxyl group of the sialic acidis protected with a protective group.

The present invention provides a process for preparing a glycopeptidewherein the asparagine-linked oligosaccharide in (6) is anasparagine-linked asialooligosaccharide.

The present invention provides a process for preparing a glycopeptidewherein a mucin-linked oligosaccharide is used in place of a portion orthe whole of the asparagine-linked oligosaccharide.

The present invention provides a glycopeptide which is obtainable by theabove processes and which has at least one asparagine-linkedoligosaccharide or mutin-linked oligosaccharide at a desired position ofthe peptide chain thereof.

The present invention provides a glycopeptide wherein theasparagine-linked oligosaccharide or the mutin-linked oligosaccharidehas at least 6 sugar residues, and has a bifurcated oligosaccharideattached thereto.

The present invention provides a glycopeptide which is a glycopeptidehaving at least one oligosaccharide selected from amongasparagine-linked disialooligosaccharide and asparagine-linkedmonosialooligosaccharide attached as the asparagine-linkedoligosaccharide.

The present invention provides a process for preparing glycopeptidehaving at least one asparagine-linked oligosaccharide at a desiredposition of the peptide chain thereof and a residue of sialic acid or aderivative thereof at a terminal end thereof, the process comprising:

(1) esterifying a hydroxyl group of a resin having the hydroxyl groupand a carboxyl group of an amino acid having amino group nitrogenprotected with a fat-soluble protective group,

(2) removing the fat-soluble protective group to form a free aminogroup,

(3) amidating the free amino group and a carboxyl group of an amino acidhaving amino group nitrogen protected with a fat-soluble protectivegroup,

(4) removing the fat-soluble protective group to form a free aminogroup,

(5) repeating the steps (3) and (4) at least once,

(6) amidating the free amino group and a carboxyl group of theasparagine portion of an asparagine-linked oligosaccharide having aminogroup nitrogen protected with a fat-soluble protective group,

(7) removing the fat-soluble protective group to form a free aminogroup,

(8) amidating the free amino group and a carboxyl group of an amino acidhaving amino group nitrogen protected with a fat-soluble protectivegroup,

(9) repeating the steps (7) and (8) at least once,

(10) removing the fat-soluble protective group to form a free aminogroup,

(11) cutting off the resin with an acid, and

(12) transferring sialic acid or a derivative thereof to the resultingglycopeptide using a sialic acid transferase.

The present invention provides a process for preparing a glycopeptidewherein a marker is reacted with the resin before the resin is cut offwith the acid in step (11).

The present invention provides a process for preparing a glycopeptidewherein the marker is a dansyl halide.

The present invention provides a process for preparing5-acetamido-3,5,7-trideoxy-7-fluoro-D-glycero-β-D-lacto-2-nonulopyranosidonicacid comprising reacting N-acetyl-4-deoxy-4-fluoro-D-mannosamine, sodiumpiruvate, bovine serum albumin and aldolase sialate.

The present invention provides a process for preparing5-acetamido-3,5,7-trideoxy-7-fluoro-D-glycero-β-D-lacto-2-nonulopyranosidonicacid comprising hydrogenating benzyl2-azido-2,4-dideoxy-4-fluoro-β-D-mannopyranoside in the presence ofacetic anhydride to obtain N-acetyl-4-deoxy-4-fluoro-D-mannosamine, andsubsequently reacting the product with sodium piruvate, bovine serumalbumin and aldolase sialate.

The present inventor has already developed, as disclosed in JapanesePatent Application No. 2001-185685 (hereinafter referred to as the“prior application”), processes for preparing asparagine-linkedoligosaccharides derivative, asparagine-linked oligosaccharides andoligosaccharides which processes are capable of producing variousisolated asparagine-linked oligosaccharides derivative with greater easeand in larger quantities than conventionally, and further novelasparagine-linked oligosaccharides derivative, asparagine-linkedoligosaccharides and oligosaccharides, wherein oligosaccharidesdeficient in sugar residues as desired are linked.

The processes of the prior application include:

(1) a process for preparing an asparagine-linked oligosaccharidederivative derived from an asparagine-linked oligosaccharide whichprocess includes the steps of:

(a) introducing a fat-soluble protective group into an asparagine-linkedoligosaccharide or at least two asparagine-linked oligosaccharidesincluded in a mixture comprising the oligosaccharide or said at leasttwo oligosaccharides to obtain an asparagine-linked oligosaccharidederivative mixture, and

(b) hydrolyzing the asparagine-linked oligosaccharide derivative mixtureor asparagine-linked oligosaccharides derivative included in thismixture and subjecting the resulting mixture to chromatography toseparate off asparagine-linked oligosaccharides derivative,

(2) a process for preparing an asparagine-linked oligosaccharidederivative according to (1) which further includes the step (b′) ofhydrolyzing the asparagine-linked oligosaccharides derivative separatedoff by the step (b) with a sugar hydrolase,(3) a process for preparing an asparagine-linked oligosaccharidederivative according to (1) or (2) wherein the mixture comprising theoligosaccharide or said at least two oligosaccharides includes acompound of the formula (A) below and/or a compound corresponding tosaid compound wherein at least one sugar residue is deficient,(4) a process for preparing an asparagine-linked oligosaccharidederivative according to any one of (1) to (3) wherein the fat-solubleprotective group is a fluorenylmethoxycarbonyl (Fmoc) group,(5) a process for preparing an asparagine-linked oligosaccharidederivative according to any one of (1) to (3) wherein the step (a) isthe step of introducing Fmoc group into the asparagine-linkedoligosaccharide or said at least two asparagine-linked oligosaccharideshaving a sialic residue at a nonreducing terminal and included in themixture, and introducing benzyl group into the sialic residue to obtainthe asparagine-linked oligosaccharide derivative mixture,(6) A process for preparing an asparagine-linked oligosaccharideincluding the steps of:

(a) introducing a fat-soluble protective group into an asparagine-linkedoligosaccharide or at least two asparagine-linked oligosaccharidesincluded in a mixture comprising the oligosaccharide or said at leasttwo oligosaccharides to obtain an asparagine-linked oligosaccharidederivative mixture,

(b) hydrolyzing the asparagine-linked oligosaccharide derivative mixtureor asparagine-linked oligosaccharides derivative included in thismixture and subjecting the resulting mixture to chromatography toseparate off asparagine-linked oligosaccharides derivative, and

(c) removing the protective group from the asparagine-linkedoligosaccharides derivative separated off in the step (b) to obtainasparagine-linked oligosaccharides,

(7) a process for preparing an asparagine-linked oligosaccharideaccording to (6) which further includes:

the step (b′) of hydrolyzing the asparagine-linked oligosaccharidesderivative separated off by the step (b) with a sugar hydrolase, and/or

the step (c′) of hydrolyzing the asparagine-linked oligosaccharidesobtained by the step (c) with a sugar hydrolase,

(8) a process for preparing an asparagine-linked oligosaccharideaccording to (6) or (7) wherein the mixture comprising theoligosaccharide or said at least two oligosaccharides includes acompound of the formula (A) below and/or a compound corresponding tosaid compound wherein at least one sugar residue is deficient,(9) a process for preparing an asparagine-linked oligosaccharideaccording to any one of (6) to (8) wherein the fat-soluble protectivegroup is Fmoc group.(10) a process for preparing an asparagine-linked oligosaccharideaccording to any one of (6) to (8) wherein the step (a) is the step ofintroducing Fmoc group into the asparagine-linked oligosaccharide orsaid at least two asparagine-linked oligosaccharides having a sialicresidue at a nonreducing terminal and included in the mixture, andintroducing benzyl group into the sialic residue to obtain theasparagine-linked oligosaccharide derivative mixture, etc.

The above asparagine-linked oligosaccharide derivative is represented,for example, by the formula (6).

wherein R¹ and R² are each a hydrogen atom or a group represented by oneof the formula (2) to (5), and may be the same or different except thatR¹ and R² are each the group of the formula (3).

Another asparagine-linked oligosaccharide derivative is represented, forexample, by the formula (7).

wherein one of R^(x) and R^(y) is a group represented by the formula(8), and the other is a hydrogen atom or a group represented by one ofthe formulae (2) to (5) and (8).

The above asparagine-linked oligosaccharide is represented, for example,by the formula (1).

wherein R³ and R⁴ are each a hydrogen atom or a group represented by oneof the formula (2) to (5), and may be the same or different except thatR³ and R⁴ are each the group of the formula (2) or the formula (3).

Since a detailed description is given in the prior application about thepreparation of these asparagine-linked oligosaccharide derivatives andasparagine-linked oligosaccharides, reference will be made to theapplication. However, what is disclosed in the prior application will bedescribed to some extent. The process of the prior application forpreparing asparagine-linked oligosaccharides derivative is distinctlycharacterized in that a fat-soluble protective group is introduced into(linked with) a asparagine-linked oligosaccharide derived from anaturally occurring glycoprotein, preferably asparagine-linkedoligosaccharides included in a mixture of asparagine-linkedoligosaccharides obtained from oligosaccharides capable of linking toasparagine, to obtain a mixture of asparagine-linked oligosaccharidesderivative, followed by separation of the mixture into individualasparagine-linked oligosaccharides derivative. The term an“asparagine-linked oligosaccharide” as used herein refers to anoligosaccharide having asparagine linked thereto. Further the term“oligosaccharides capable of linking to asparagine” refers to a group ofoligosaccharides wherein N-acetylglucosamine present at a reducingterminal is attached by N-glucoside linkage to the acid amino group ofasparagine (Asn) in the polypeptide of a protein and which hasMan(β1-4)GlcNac(β1-4)GlcNac as the mother nucleus. The term an“asparagine-linked oligosaccharide derivative” refers to anasparagine-linked oligosaccharide wherein a fat-soluble protective groupis attached to asparagine residue. Further “AcHN” in the structuralformulae of compounds refers to an acetamido group.

As described previously, oligosaccharides derived from naturallyoccurring glycoproteins are a mixture of oligosaccharides which arerandomly deficient in the sugar residue at the nonreducing terminal. Thepresent inventors have unexpectedly found that the introduction of afat-soluble protective group into an oligosaccharide derived from anaturally occurring glycoprotein, preferably into asparagine-linkedoligosaccharides included in a mixture thereof, makes it possible toreadily separate a mixture of asparagine-linked oligosaccharidesderivative having the protective group introduced therein intoindividual asparagine-linked oligosaccharides derivative by a knownchromatographic procedure. Consequently, asparagine-linkedoligosaccharides derivative having different structures can be preparedindividually in large quantities. For example, asparagine-linkedoligosaccharides derivative which resemble in structure and which areconventionally difficult to separate can be separated from one another,and these compounds can be prepared easily in large quantities. Furthera sugar hydrolase can be caused to act on the resultingasparagine-linked oligosaccharides derivative and thereby preparevarious asparagine-linked oligosaccharides derivative.

Thus, introducing a fat-soluble protective group into asparagine-linkedoligosaccharides provides derivatives and makes it possible to separatethe asparagine-linked oligosaccharides derivative from one another.Presumably this is attributable to the fact that the introduction of thefat-soluble protective group gives improved fat solubility to the wholeasparagine-linked oligosaccharides derivative to ensure remarkablyimproved interaction between the oligosaccharide and the reverse-phasecolumn to be used favorably, consequently separating theasparagine-linked oligosaccharides derivative from one another byreflecting the difference of structure between the oligosaccharides withhigh sensitivity.

Further by removing the protective group from the asparagine-linkedoligosaccharides derivative obtained, various asparagine-linkedoligosaccharides can be artificially prepared easily in large amountsaccording to the prior application.

The process of the present invention provides the desired glycopeptidesusing various asparagine-linked oligosaccharides obtained by the priorapplication.

In the process of the present invention, (1) subjected to an esterifyingreaction are a hydroxyl group of a resin having the hydroxyl group and acarboxyl group of an amino acid having amino group nitrogen protectedwith a fat-soluble protective group.

Since the amino group nitrogen of the amino acid is protected with afat-soluble protective group, the hydroxyl group of the resin is reactedwith the carboxyl group of the amino acid, with self-condensation of theamino acid prevented.

Next, (2) the fat-soluble protective group is removed from the resultingester to form a free amino group,

(3) the free amino group is amidated with a carboxyl group of a desiredamino acid having amino group nitrogen protected with a fat-solubleprotective group,

(4) the fat-soluble protective group is removed to form a free aminogroup, and

(5) the steps (3) and (4) are repeated at least once to thereby obtain apeptide having a desired number of desired amino acids as linked andhaving the resin attached to one end thereof and a free amino group atthe other end thereof.

Next, (6) the free amino group is amidated with a carboxyl group of theasparagine portion of an asparagine-linked oligosaccharide having aminogroup nitrogen protected with a fat-soluble protective group,

(7) the fat-soluble protective group is removed to form a free aminogroup,

(8) the free amino group is amidated with a carboxyl group of a desiredamino acid having amino group nitrogen protected with a fat-solubleprotective group,

(9) the steps (7) and (8) are repeated at least once, and

(10) the fat-soluble protective group is removed to form a free aminogroup and thereby obtain a glycopeptide having a desired number ofdesired amino acids as linked and having the resin attached to one endthereof, a free amino group at the other end thereof and anasparagine-linked oligosaccharide at an intermediate position.

(11) The resin is cut off with an acid, whereby a glycopeptide can beprepared which has an asparagine-linked oligosaccharide at a desiredposition of the peptide chain thereof.

Furthermore, a glycopeptide having at least two asparagine-linkedoligosaccharides at a desired position of the peptide chain thereof canbe prepared by suitably adding the step (6) of amidating the free aminogroup and a carboxyl group of the asparagine portion of anasparagine-linked oligosaccharide having amino group nitrogen protectedwith a fat-soluble protective group. At this time, a glycopeptide havingat least two kinds of asparagine-linked oligosaccharides at a desiredposition of the peptide chain thereof can be prepared by using adifferent asparagine-linked oligosaccharide.

Alternatively, the asparagine-linked oligosaccharide can be introducedinto an end portion of the peptide chain.

Furthermore, a mucin-linked oligosaccharide can be used in place of aportion or whole of the asparagine-linked oligosaccharide.

The resin having a hydroxyl group for use in the present invention mayusually be a resin having hydroxyl useful for solid-phase synthesis.Examples of resins usable are Wang resin (product of Merk), HMPA-PEGAresin (product of Merk), etc.

All amino acids are usable as such. Examples of amino acids usable areserine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine(Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys),arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid(Glu), glutamine (Gln), threonine (Thr), cysteine (Cys), methionine(Met), phenylalanine (Phe) tryptophan (Trp) and proline (Pro).

Examples of fat-soluble protective groups are 9-fluorenylmethoxycarbonyl(Fmoc) group, tert-butyloxycarbonyl (Boc) group, benzyl group, allylgroup, allyloxycarbonyl group, acetyl group and like carbonate-type oramide-type protective groups. The fat-soluble protective group, e.g.,Fmoc group, can be introduced by adding 9-fluorenylmethyl-N-succinimidylcarbonate and sodium hydrogencarbonate to the contemplated compound forreaction. The reaction is conducted at 0 to 50° C., preferably at roomtemperature, for about 1 to about 5 hours.

The above amino acid can be protected with a fat-soluble protectivegroup by the method described above. The above protected amino acid canbe those available commercially. Examples are Fmoc-Ser, Fmoc-Asn,Fmoc-Val, Fmoc-Leu, Fmoc-Ile, Fmoc-Ala, Fmoc-Tyr, Fmoc-Gly, Fmoc-Lys,Fmoc-Arg, Fmoc-His, Fmoc-Asp, Fmoc-Glu, Fmoc-Gln, Fmoc-Thr, Fmoc-Cys,Fmoc-Met, Fmoc-Phe, Fmoc-Trp and Fmoc-Pro.

Usable as esterifying catalysts are dehydrating condensation agents suchas 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT),dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIPCDI). Theesterifying reaction is conducted preferably by placing a resin, forexample, into a solid-phase column, washing the resin with a solvent andthereafter adding a solution of amino acid in a solvent to the resin.Examples of solvents for washing are dimethylformamide (DMF),2-propanol, methylene chloride, etc. Examples of solvents for dissolvingamino acids are dimethyl sulfoxide (DMSO), DMF, methylene chloride, etc.The reaction is conducted at 0 to 50° C., preferably at roomtemperature, for about 10 to about 30 hours, preferably about 15 minutesto about 24 hours.

Preferably, the unreacted hydroxyl group remaining on the solid phase atthis time is acetylated, for example, with acetic anhydride for capping.

The fat-soluble protective group can be removed, for example, by atreatment with a base. Examples of bases to be used are piperidine,morpholine, etc. This treatment is conducted preferably in the presenceof a solvent. Examples of solvents usable are DMSO, DMF, methanol, etc.

The reaction of amidating the free amino group with a carboxyl group ofa desired amino acid having amino group nitrogen protected with thefat-soluble group is conducted, preferably in the presence of anactivator and a solvent.

Examples of useful activators are dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC/HCl),diphenylphosphorylazide (DPPA), carbonyldiimidazole (CDI),diethylcyanophosphonate (DEPC),benzotriazole-1-yloxy-trispyrrolidinophosphonium (DIPCI),benzotriazole-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate(PyBOP), 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu),dimethylaminopyridine (DMAP), 1-hydroxy-7-azabenzotriazole (HOAt),hydroxyphthalimide (HOPht), pentafluorophenol (Pfp-OH),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphonate (HATU),O-benzotriazole-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), 3,4-dihydro-3-hydrodi-4-oxa-1,2,3-benzotriazine (Dhbt).

The activator is used in an amount of 1 to 20 equivalents, preferably 1to 10 equivalents, more preferably 1 to 5 equivalents, based on an aminoacid having amino group nitrogen protected with a fat-soluble protectivegroup.

Examples of useful solvents are DMSO, DMF, methylene chloride, etc. Itis desired that the reaction be conducted at 0 to 50° C., preferably atroom temperature, for about 10 to about 30 hours, preferably about 15minutes to about 24 hours. It is desired that the unreacted hydroxylgroup remaining on the solid phase at this time be acetylated, forexample, with acetic anhydride for capping. The fat-soluble protectivegroup can be removed in the same manner as described above.

The peptide chain is cut off from the resin, preferably by a treatmentwith an acid. Examples of acids to be used are trifluoroacetic acid(TFA), hydrogen fluoride (HF), etc.

According to the invention, a glycopeptide having at least twoasparagine-linked oligosaccharides at a desired position of the peptidechain thereof can be prepared by suitably additionally performing thesteps (6) of amidating the free amino group and a carboxyl group of theasparagine portion of an asparagine-linked oligosaccharide having aminogroup nitrogen protected with a fat-soluble protective group, and (7) ofremoving the fat-soluble protective group to form a free amino group.

Further according to the invention, a glycopeptide having at least oneasparagine-linked oligosaccharide at a desired position of the peptidechain thereof can be prepared by performing as final steps the steps (6)of amidating the free amino group and the carboxyl group of theasparagine portion of an asparagine-linked oligosaccharide having aminogroup nitrogen protected with a fat-soluble protective group, and (7) ofremoving the fat-soluble protective group to form a free amino group.

Further according to the invention, a glycopeptide having anasparagine-linked oligosaccharide at an end portion can be prepared byperforming the step (1) of esterifying a hydroxyl group of a resinhaving the hydroxyl group and a carboxyl group of an amino acid havingamino group nitrogen protected with a fat-soluble protective group, inplace of the step (6) or in addition to the step (6).

The asparagine-linked oligosaccharides to be used in the presentinvention can be those having a desired number of sugar residues. Anasparagine-linked oligosaccharide or mucin-linked oligosaccharide isespecially usable which has at least six sugar residues and which hasnot been used conventionally. This is a unique feature of the invention.It is also possible to use asparagine-linked oligosaccharides having 9to 11 sugar residues.

It is further possible to use asparagine-linked oligosaccharides of thebifurcated type which has at least six sugar residues. For example, theasparagine-linked oligosaccharide to be used can be an asparagine-linkeddisialooligosaccharide or asparagine-linked monosialooligosaccharide.Glycopeptides incorporating such an asparagine-linkeddisialooligosaccharide or asparagine-linked monosialooligosaccharide arepreferred glycopeptides of the invention.

The asparagine-linked oligosaccharide to be used can be anasparagine-linked disialooligosaccharide or asparagine-linkedmonosialooligosaccharide wherein the carboxyl group of sialic acid isprotected with a protective group.

In the case of the asparagine-linked oligosaccharide or mucin-linkedoligosaccharide for use in the invention, the oligosaccharide may haveits hydroxyl protected. Examples of protective groups useable areacetyl, triethylsilyl, etc. Preferably, the protective group is onewhich can be treated with an acid simultaneously when the resin is cutoff from the glycopeptide prepared. For example, triethylsilyl is usefulas such.

In the case where the asparagine-linked oligosaccharide is anasparagine-linked disialooligosaccharide or asparagine-linkedmonosialooligosaccharide, there is a likelihood that the sialic acidwill be cut off with an acid, so that such oligosaccharides wherein thecarboxyl group of sialic acid is protected with a protective group aredesirable since the sialic acid is then prevented from being cut off.Examples of protective groups to be used are benzyl, allyl,diphenylmethyl group, etc.

The reaction for introducing a protective group into the carboxyl groupof sialic acid can be conducted in a known manner, for example, asdisclosed in “Protective Groups in Organic Chemistry,” John Wiley & SonsINC., New York 1991, ISBN 0-471-62301-6.

According to the invention, derivatives of sialic acid are those whereinthe hydroxyl group attached to the carbon atom at the 7-position,8-position or 9-position of the sialic acid is replaced by a hydrogenatom or halogen atom. Examples of halogen atoms are fluorine, chlorine,bromine and the like, among which fluorine is preferred.

The sialic acid transferase to be used in the present invention can bethose generally available commercially. A suitable transferase isselectable in accordance with the kind of contemplated sialic acid orsialic acid derivative and the mode of linkage. Examples of usefultransferases are those derived from a rat recombinant and rat liver.Sialytase may be used for pH adjustment to shift the equilibrium andeffect a transfer reaction for sialic acid or a derivative thereof.

The glycopeptides of the invention are very useful in the field ofdevelopment of pharmaceuticals. For example, vaccines for cancers are anexample of application to the development of drugs. It is known thatcells developing cancer produce an oligosaccharide which is not found inthe living body. It is also known that when chemically prepared andgiven to the human body as a vaccine, such an oligosaccharide inhibitsthe growth of cancer. If the desired glycopeptide can be producedaccording to the invention, it is possible to prepare a vaccine which iseffective for treating cancer. The glycopeptide obtained by theinvention can further be made into derivatives by attaching novel sugarresidues thereto through combinations of chemical reactions andreactions of sugar transferases for the preparation of novel vaccines.

Glycopeptides exhibit higher solubility in water than peptides which arenot combined with oligosaccharides, while they are highly stable when inthe form of aqueous solutions and when present in blood.

The sialic acid at the nonreducing terminal, when made into aderivative, prevents the decomposition of the oligosaccharide itself,thereby giving enhanced stability to the glycopeptide.

Furthermore, the sialic acid at the nonreducing terminal, as made into aderivative is a nonnatural-type oligosaccharide and can therefore beeffective for the preparation of vaccines.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention will be described below with reference toexamples, to which the invention is not limited.

Used in the following examples are Fmoc-Val, Fmoc-Leu, Fmoc-Leu-Opfp,Fmoc-Ala, Fmoc-Ala-Opfp, Fmoc-Val-Opfp, Fmoc-Ser(Bzl)-OH andFmoc-Ser(OtBu) which are known substances. Commercial products are usedas these substances. For example, Opfp in Leu-Opfp stands for leucine(Leu) having the carboxyl group thereof protected with pentafluorophenyl(pfp), Ser(Bzl)-OH for serine (Ser) having the hydroxyl thereofprotected with benzyl (Bzl), and Ser(OtBu)-OH for serine (Ser) havingthe hydroxyl thereof protected with t-butyl (tBu).

Reference Example 1 Preparation of Asparagine-LinkedDisialooligosaccharide (10)

A 500 mg quantity of roughly purified SGP (sialylglycopeptide) and 10 mg(319 μmols) of sodium azide were dissolved in 25 ml of tris-hydrochloricacid-calcium chloride buffer solution (0.05 mol/l of TRIZMA BASE, 0.01mol/l of calcium chloride, pH=7.5). To the solution was added a solutionof 50 mg of actinase E (protease, product of Kaken Seiyaku) in 5 ml oftris-hydrochloric acid-calcium chloride buffer solution, followed bystanding at 37° C. The solution was freeze-dried 115 hours later. Theresidue was purified by gel filtration column chromatography twice,giving 252 mg of the desired product, i.e., Asparagine-linkeddisialooligosaccharide (10).

¹H-NMR (30° C.) δ 5.13 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.5 Hz,GlcNAc1-H-1), 4.95 (s, 1H, Man4-H-1), 4.77 (s, 1H, Man3-H-1), 4.61 (d,1H, J=7.6 Hz, GlcNAc2-H-1), 4.60 (d, 2H, J=7.6 Hz, GlcNAc5, 5-H-1), 4.44(d, 2H, J=8.0 Hz, Gal6, 6-H-1), 4.25 (bd, 1H, Man3-H-2), 4.20 (bdd, 1H,Man4-H-2), 4.12 (bd, 1H, Man4-H-2), 2.94 (dd, 1H, J=4.5 Hz, 17.2 Hz,Asn-βCH), 2.85 (dd, 1H, J=7.0 Hz, 17.2 Hz, Asn-βCH), 2.67, 2.66 (dd, 2H,J=4.6 Hz, 12.4 Hz, NeuAc7, 7-H-3_(eq)), 2.07 (s, 3H, Ac), 2.06 (s, 6H,Ac×2), 2.02 (s, 6H, Ac×2), 2.01 (s, 3H, Ac), 1.71 (dd, 2H, J=12.4 Hz,12.4 Hz, NeuAc7, 7-H-3_(ax).

Reference Example 2 Preparation of Asparagine-LinkedDisialooligosaccharide (11) Wherein Amino Group Nitrogen of Asparagineis Protected with Fmoc Group

An 80 mg quantity (0.034 mmol) of the asparagine-linkeddisialooligosaccharide obtained in Reference Example 1 was dissolved ina solution of 2.7 ml of distilled water and 4.1 ml of acetone, and tothe solution were added 34.7 mg (0.103 mmol) of9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSn) and 11.5 mg (0.137mmol) of sodium hydrogencarbonate. The mixture was stirred at roomtemperature for 2 hours. After the completion of reaction was recognizedby TLC, the resulting solution was concentrated in a vacuum to removeacetone. The residue was applied to a column (ODS column) filled with asilica gel having octadecylsilyl group attached thereto) forpurification, affording 60.1 mg of the desired product, i.e.,Fmoc-asparagine-linked disialooligosaccharide (11) in a yield of 68%.

¹H-NMR (30° C.) 8.01 (2H, d, J=7.5 Hz, Fmoc), 7.80 (2H, d, J=7.5 Hz,Fmoc), 7.60 (2H, dd, J=7.5 Hz, Fmoc), 7.53 (2H, dd, J=7.5 Hz, Fmoc),5.23 (1H, s, Man4-H₁), 5.09 (1H, d, J=9.4 Hz, GlcNAc1-H₁), 5.04 (1H, s,Man4′-H₁), 4.86 (1H, s, Man3-H₁), 4.70˜4.66 (m, GlcNAc2-H₁ GlcNAc5,5′-H₁), 4.54 (2H, d, J=7.9 Hz, Gal6, 6′-H₁), 4.44 (1H, d, FmocCH), 4.34(1H, bd, Man3-H₂), 4.29, (1H, bd, Man4′-H₂), 4.20 (1H, bd, Man4-H₂),2.77 (2H, dd, NeuAc7, 7′-H_(3eq)), 2.80 (1H, bdd, Asn-βCH), 2.62 (1H,bdd, Asn-βCH), 2.14 (18H, s×6, -Ac), 1.80 (2H, dd, NeuAc7, 7-H_(3ax))

Reference Example 3 Preparation of Asparagine-LinkedDisialooligosaccharide (12) Wherein Amino Group Nitrogen of Asparagineis Protected with Fmoc Group, and Carboxyl Group of Sialic Acid isProtected with Benzyl Group

A cold aqueous solution of Fmoc-asparagine-linked bifurcateddisialooligosaccharide (20 mg) was passed through a column [φ0.5 cm×5cm] of Dowex-50Wx8(H⁺), and the eluate of aqueous solution wasfreeze-dried.

The Fmoc-asparagine-linked bifurcated disialooligosaccharide obtainedwas dissolved in cold water at 4° C., an aqueous solution of Cs₂CO₃ (2.5mg/ml) was added to the solution to obtain an adjusted pH of 5 to 6, andthe oligosaccharide solution was freeze-dried. The resulting sample ofFmoc-disialooligosaccharide was dissolved in dry DMF (1.3 ml), benzylbromide (5.1 μl) was added to the solution, and the mixture was stirredat room temperature under an argon stream for 45 hours. After thecompletion of reaction was recognized by TLC, the reaction mixture wascooled to 0° C., and 10 ml of diethyl ether was added to the mixture toseparate out the desired product. The product was filtered with filterpaper. Distilled water was added to the remaining desired product, and afiltrate was obtained from the mixture and subsequently concentrated ina vacuum. The residue obtained was purified by an ODS column to obtain18.2 mg (85% in yield) of the desired product, i.e.,Fmoc-asparagine-linked disialooligosaccharide (12).

¹H-NMR (30° C.), 7.90 (d, 2H, Fmoc), 7.70 (d, 2H, Fmoc), 7.53-7.40 (m,9H, Bn, Fmoc), 5.36 (d, 2H, J=11.6, Hz, CH₂), 5.30 (d, 2H, J=11.6 Hz,CH₂), 5.12 (s, 1H, Man4-H₁), 4.99 (d, 1H, J=9.7 Hz, GlcNAc1-H₁), 4.93(s, 1H, Man4′-H₁), 4.75 (s, 1H, Man3-H₁), 4.57 (m, 3H, GlcNAc2-H₁,GlcNAc5, 5′-H₁), 4.32 (d, 2H, Gal6, 6′-H₁), 4.24 (d, 1H, Man3-H₂), 4.18(d, 1H, Man4′-H₂), 4.10 (1H, d, Man4-H₂), 2.72 (bd, 1H, Asn-βCH), 2.67(dd, 2H, NeuAc7, 7′-H_(3eq)), 2.51 (bdd, 1H, Asn-βCH), 2.06 (s, 3H, Ac),2.03, 2.01 (each s, each 6H, Ac×2), 1.89 (s, 3H, Ac), 1.83 (2H, dd,J=12.2, 12.2 Hz, NeuAc7, 7′-H_(3ax))

HRMS Calcd for C₁₁₇H₁₆₅N₈Na₂O₆₆[M+Na⁺] 2783.9597, found 2783.9501

Reference Example 4

Asparagine-linked monosialooligosaccharide was prepared according toJapanese Patent Application No. 2001-185685.

Reference Example 5 Preparation of HOOC-Val-Leu-Leu-Ala-NH₂ (13)

I: Introduction into Resin

Wang resin (1.6 g) was placed into a solid-phase synthesis column, andthe resin was fully washed with methylene chloride and then withmethanol and dried. A 409.2 mg quantity (1.2 mmols) of Fmoc-Val and121.5 mg (0.9 mmol) of 1-hydroxybenzotriazole hydrate (HOBt.H₂O) weredissolved in 4.5 ml of N,N-dimethylacetamide (DMA), 247.5 mg (1.2 mmols)of dicyclohexylcarbodiimide (DCC) was added to the solution, and themixture was stirred at 0° C. for 15 minutes to obtain an amino acidsolution. The resin was swollen with DMF. The amino acid solution wasplaced into the solid-phase synthesis column and stirred at roomtemperature for 17 hours. The resin was thereafter washed with methylenechloride, then with isopropanol and thereafter with methanol, and dried.

The dried resin was swollen with DMF in a column, about 10 ml of a 20%piperidine/DMF solution was thereafter added to the resin, followed bystirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Val-NH₂. The resin was then washed with DMFand dried.

II: Lengthening Peptide Chain

The dried resin (resin-Val-NH₂) was swollen with DMF in a column, 318.6mg (0.9 mmol) of Fmoc-Leu and 121.5 mg (0.9 mmol) of HOBt.H₂O werethereafter added to the resin, and DMF was further added in an amount toimmerse the resin. With addition of 138.5 μl (0.9 mmol) ofdiisopropylcarbodiimide (DIPCDI), the mixture was stirred at roomtemperature for 2 hours. The resin was thereafter washed with DMF anddried.

The dried resin was swollen with DMF in a column, about 10 ml of a 20%piperidine/DMF solution was thereafter added to the resin, followed bystirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Val-Leu-NH₂. The resin was then washed withDMF and dried.

The dried resin was swollen with DMF in a column, 318.6 mg (0.9 mmol) ofFmoc-Leu and 121.5 mg (0.9 mmol) of HOBt.H₂O were added to the resin,and DMF was further added in an amount to immerse the resin. Withaddition of 138.5 μl (0.9 mmol) of DIPCDI, the mixture was stirred atroom temperature for 2 hours. The resin was thereafter washed with DMFand dried.

The dried resin was swollen with DMF in a column, about 10 ml of a 20%piperidine/DMF solution was thereafter added to the resin, followed bystirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Val-Leu-Leu-NH₂. The resin was then washedwith DMF and dried.

The dried resin was swollen with DMF in a column, 293.4 mg (0.9 mmol) ofFmoc-Ala and 121.5 mg (0.9 mmol) of HOBt.H₂O were added to the resin,and DMF was further added in an amount to immerse the resin. Withaddition of 138.5 μl (0.9 mmol) of DIPCDI, the mixture was stirred atroom temperature for 2 hours. The resin was thereafter washed with DMFand dried.

The dried resin was swollen with DMF in a column, about 10 ml of a 20%piperidine/DMF solution was added to the resin, followed by stirring atroom temperature for 15 minutes to obtain resin-Val-Leu-Leu-Ala-NH₂ byremoving the protective Fmoc group. The resin was then washed with DMFand dried.

III: Separation from Resin

Preparation of HOOC-Val-Leu-Leu-Ala-NH₂

A 5% aqueous solution of TFA was added to the dried resin, and themixture was stirred at room temperature for 3 hours. The solution wasthereafter transferred to an egg-shaped flask, and diethyl ether wasadded to the solution with the flask placed in ice to precipitate thedesired product, followed by filtration.

¹H-NMR (30° C.) 8.56 (1H, d, J=6.5 Hz, Leu-2NH), 8.42 (1H, d, J=7.4 Hz,Leu-1NH), 8.25 (1H, d, J=8.3 Hz, Val NH), 4.34 (1H, d, J=6.7 Hz, Val-α),4.16 (1H, d, J=7.1 Hz, Ala-α), 2.27 (1H, ddd, Val-β), 1.69˜1.58 (m, 11H,Leu-1, Leu-2), 1.59 (3H, d, J=7.2 Hz, Ala-β), 1.01˜0.96 (m, 25H, Leu-1,Leu-2, Val)

Example 1

The resin (17 mg) in the form of dry resin-Val-Leu-Leu-Ala-NH₂ preparedin Reference Example 4 and before separation from the solid phase isplaced into Eppen tube. A 35 mg quantity (14.8 μmols) ofDibenzyl-Fmoc-asparagine-linked disialooligosaccharide (12) obtained inReference Example 3 and 0.64 mg (2.7 μmols) ofO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphonate (HATU) were added to the resin, and 150 μl of DMFwas added. With addition of 0.31 μl of diisopropylethylamine (DIPEA),the mixture was stirred at room temperature for 24 hours. The resultingmixture was thereafter washed with DMF and dried.

The dried resin was swollen with DMF in an Eppen tube, about 1 ml of a20% piperidine/DMF solution was thereafter added to the resin, followedby stirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Val-Leu-Leu-Ala-Asn(oligo)-NH₂. The resinwas then washed with DMF and dried. Asn(oligo) mentioned stands fordibenzyl-asparagine-linked disialooligosaccharide obtained by removingthe Fmoc group from the Dibenzyl-Fmoc-asparagine-linkeddisialooligosaccharide (12) obtained in Reference Example 3.

(Separation from Solid Phase)

Preparation of HOOC-Val-Leu-Leu-Ala-Asn(oligo)-NH₂

An aqueous solution (95%) of trifluoroacetic acid (TFA) was added to theabove dried resin, followed by stirring at room temperature for 3 hours.The solution was thereafter transferred to an Eppen tube, diethyl etherwas added to the solution with the tube placed in ice to precipitate thedesired product. The precipitate was dissolved in 0.1% aqueous solutionof TFA, and the solution was purified by a reverse phase columnchromatography. (YMC-Pack ODS-A 250×3.0 mm, flow rate 0.45 ml/min,developing solvent A: 0.1% TFA aqueous solution, B: 0.1% TFAacetonitrile:water=90:10, gradient only A 10 min, A100%→B100% 30 min).

The structure of Asn(oligo) is shown below.

¹H-NMR (30° C.) 7.59˜7.55 (m, 10H, Bn), 5.45 (4H, dd, Bn-CH₂×2), 5.23(1H, s, Man4-H₁), 5.15 (1H, d, GlcNAc1-H₁), 5.03 (1H, s, Man4′-H₁), 4.87(1H, s, Man3-H₁), 4.67 (3H, d, GlcNAc2-H₁, GlcNAc5, 5′-H₁), 4.42 (2H, d,Gal6, 6′-H₁), 4.34 (1H, d, Man3-H₂), 4.28 (1H, d, Man4′-H₂), 4.20 (1H,d, Man4-H₂), 2.82 (2H, dd, J=6.68 Hz, NeuAc7, 7′-H_(3eq)), 2.65 (1H, dd,J=16.69 Hz, 6.83 Hz, Asn-βCH), 2.13 (18H, s×6, -Ac), 1.94 (2H, dd,J=12.24 Hz, NeuAc7, 7′-H_(3ax)), 1.41˜1.26 (m, 25H, Leu-1, Leu-2, Val)

Example 2 Preparation of HOOC-Ser-Ser-Asn(oligo)-NH₂

I: Introduction into Resin

PEGA resin (50 mg) was placed into a solid phase synthesis column andthoroughly washed with methylene chloride and then with methanol anddried.

An 80 mg quantity (180 μmols) of Fmoc-Ser(Bzl)-OH and 19 mg (135 μmols)of HOBt.H₂O were dissolved in 10 ml of DMF, 37 mg (180 μmols) of DCC wasadded to the solution, and the mixture was stirred at 0° C. for 15minutes to obtain an amino acid solution. A resin was swollen with DMF.The amino acid solution was placed into a solid-phase synthesis column,followed by stirring at room temperature for 17 hours. The resin wasthereafter washed with methylene chloride, then with isopropanol andthereafter with methanol, and dried.

The dried resin was swollen with DMF in a column, about 2.0 ml of a 20%piperidine/DMF solution was thereafter added to the resin, followed bystirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Ser-NH₂. The resin was then washed withisopropanol and DMF and dried.

II: Lengthening Peptide Chain

The dried resin was swollen with DMF in a column, 40.0 mg (89.8 μmols)of Fmoc-Ser(Bzl)-OH and 12 mg (89.8 μmols) of HOBt.H₂O were thereafteradded to the resin, and 2.0 ml of DMF was further added. With additionof 14 μl (89.8 μmols) of DIPCDI, the mixture was stirred at roomtemperature for 2 hours. The resin was thereafter washed with DMF anddried.

The dried resin was swollen with DMF in a column, about 2.0 ml of a 20%piperidine/DMF solution was thereafter added to the resin, followed bystirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Ser-Ser-NH₂. The resin was then washed withisopropanol and DMF and dried.

The dried resin was swollen with dimethyl sulfoxide (DMSO) in a column,13.5 mg (5.7 μmols) of Dibenzyl-Fmoc-asparagine-linkeddisialooligosaccharide (12) obtained in Reference Example 3, asdissolved in DMF, was transferred to the column. To the mixture wereadded 1.6 mg (6.8 μmols) of HATU and 0.83 μl of DIPEA, and the resultingmixture was stirred at room temperature for 24 hours. The resin wasthereafter washed with isopropanol and DMF and dried.

The dried resin was swollen in an Eppen tube, about 1 ml of a 20%piperidine/DMF solution was thereafter added to the resin, followed bystirring at room temperature for 15 minutes to remove the protectiveFmoc group and obtain resin-Val-Ser-Ser-Asn(oligo)-NH₂. The resin wasthen washed with DMF and dried. The above Asn(oligo) was the same asthat in Example 1.

III: Separation from Solid Phase

A 95% TFA aqueous solution was added to the dried resin, followed bystirring at room temperature for 3 hours. The reaction mixture waspurified by reverse phase column chromatography. (YMC-Pack ODS-A 250×3.0mm, flow rate 0.35 ml/min, developing solvents A: 0.1% TFA aqueoussolution, B: 0.1% TFA acetonitrile:water=90:10, gradient A 100%→B 100%120 min).

¹H-NMR (30° C.) 7.60˜7.45 (m, 20H, Bn), 5.35 (4H, dd, J=11.8 Hz,Bn-CH₂—), 5.21 (1H, s, Man4-H₁), 5.13 (1H, d, J=9.2 Hz, GlcNAc1-H₁),5.03 (1H, s, Man4′-H₁), 4.34 (1H, d, Man3-H₂), 4.28 (1H, d, Man4′-H₂),4.20 (1H, d, Man4-H₂), 2.82 (2H, dd, J=6.68 Hz, NeuAc7, 7′-H_(3eq)),2.13 (18H, s×6, -Ac), 1.93 (2H, dd, J=12.24 Hz, NeuAc7, 7′-H_(3ax))

Example 3 Preparation ofHOOC-Ser-Ser-Asn(disialooligo)-Val-Leu-Leu-Ala-NH₂

Asn(disialooligo) in the desired glycopeptide mentioned above means adisialooligoasparagine having sialic acid not protected with benzylgroup.

Into a solid-phase synthesis column was placed 50 mg of HMPA-PEGA resin,which was thoroughly washed with CH₂Cl₂ and DMF.

Fmoc-Ser(OtBu)-OH, 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT)and N-methylimidazole were dissolved in CH₂Cl₂, and the solution wasstirred for 5 minutes and thereafter placed into the solid-phasesynthesis column containing the resin, followed by stirring at roomtemperature for 3 hours. The resin was thereafter washed with methylenechloride, isopropanol and DMF and dried. The unreacted amino ? group onthe solid phase was thereafter acetylated using a 20%-DMF solution ofacetic anhydride for 20 minutes for capping. The resin was washed withDMF and stirred along with a 20% piperidine/DMF solution for 20 minutesto remove the protective Fmoc group, whereby resin-Ser-NH₂ was obtained.The product was washed with DMF and dried.

Next, Fmoc-Ser(OtBu)-OH was used with HOBt.H₂O and DIPCDI forcondensation.

Subsequently, Dibenzyl-Fmoc-asparagine-linked disialooligosaccharide(12) obtained in Reference Example 3 was dissolved in a 1:1 solventmixture of DMSO and DMF, and the solution, HATU and DIPEA were stirredat room temperature for 24 hours for condensation. The resulting resinwas washed with DMF and thereafter stirred along with 10% aceticanhydride/2-propanol:methanol=: for 20 minutes for capping. The resinwas washed with 2-propanol and DMF, and thereafter stirred along with20% piperidine/DMF for 20 minutes to remove the protective Fmoc group.The resin was washed with DMF.

The resulting resin, and valine (Val), leucine (Leu), leucine (Leu) andalanine (Ala) were similarly subjected to condensation, followed byremoval of the protective Fmoc group to obtainresin-Ser-Ser-Asn(dibenzyldisialooligo)-Val-Leu-Leu-Ala-NH₂.Asn(dibenzyldisialooligo) mentioned means a disialooligoasparaginehaving sialic acid protected with benzyl group.

Used as the amino acids of valine (Val), leucine (Leu), and alanine(Ala) were each Fmoc-AA-Opfp (AA=amino acid) wherein the carboxyl groupwas pfp-esterified, and 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (Dhbt)was used for condensation. All condensation reactions were conducted ina DMF solution.

The resin resulting from condensation was thoroughly dried, andthereafter stirred along with a 95% aqueous solution of TFA at roomtemperature for 3 hours to cut off the resin. The resin was filteredoff. The reaction mixture was concentrated in a vacuum at roomtemperature, thereafter dissolved in water and freeze-dried. Theresulting product was dissolved in an aqueous solution of sodiumhydroxide having a pH of 11 to hydrolyze the benzyl ester for theremoval of benzyl group, followed by neutralization with acetic acid.The product was freeze-dried as it was, and purified by HPLC to obtainthe desired product, i.e.,HOOC-Ser-Ser-Asn(disialooligo)-Val-Leu-Leu-Ala-NH₂.

(YMC-Pack ODS-A 250×3.0 mm, developing solvents A: 0.1% TFA aqueoussolution, B: 0.1% TFA acetonitrile:water=90:10, gradient A 100% 0.35ml/min→B 100% 0.40 ml/min 90 min, flow rate 0.35 ml/min to 0.40 ml/min).

¹H-NMR (30° C.) δ 5.22 (s, 1H, Man4-H1), 5.11 (d, 1H, GlcNAc1-H1), 5.04(s, 1H, Man4′-H1), 4.86 (1H, Asnα), 4.70 (bd, 3H, GlcNAc2, 5, 5′-H1),4.62-4.57 (m, 2H, Serα×2), 4.53 (d, 2H, Gal6, 6′-H1), 4.52-4.48 (m, 2H,Leuα×2), 4.34 (bs, 1H, Man3-H2), 4.28 (bs, 1H, Man-4-H2), 4.21-4.15 (m,3H, Man4′-H2, Valα, Alaα), 2.98 (dd, 1H, Asnβ), 2.86 (dd, 1H, Asnβ),2.75 (bdd, 2H, NeuAc7, 7′-H3eq), 2.16-2.10 (Ac×6, Valβ), 1.82 (dd, 2H,NeuAc7, 7, —H3ax), 1.76-1.68 (bd, 6H, LeuβCH₂×2, LeuγCH×2), 1.60 (d, 3H,AlaβCH₃), 1.03-0.97 (m, 18H, Leu-CH₃×4, Val-CH₃×2)

Example 4 Preparation ofHOOC-Ser-Ser-Asn(Disialooligo)-Val-Leu-Leu-Ala-Asn(asialooligo)-Val-Leu-Leu-Ala-NH₂

Asn(asialooligo) in the desired glycopeptide above is anasparagine-linked oligosaccharide shown below.

Resin-Ser-Ser-Asn(dibenzyldisialooligo)-Val-Leu-Leu-Ala-NH₂ before beingseparated from the solid phase, and Asn(asialooligo), valine (Val),leucine (Leu), leucine (Leu) and alanine (Ala), were subjected tocondensation. The resulting peptide chain was cut off from the solidphase in the same manner as in Example 3, followed by the removal of thebenzyl group, affording a glycopeptide in the form ofHOOC-Ser-Ser-Asn(disialooligo)-Val-Leu-Leu-Ala-Asn(asialooligo)-Val-Leu-Leu-Ala-NH₂.

Used as the amino acids of valine (Val), leucine (Leu) and alanine (Ala)were each Fmoc-AA-Opfp (AA=amino acid) wherein the carboxyl group waspfp-esterified, and 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (Dhbt) wasused for condensation. All condensation reactions were conducted in aDMF solution. The resulting resin was stirred at room temperature for 20minutes with addition of a 20% piperidine/DMF solution to remove theprotective Fmoc group.

After the introduction of the amino acid (Val) to be positionedsubsequent to the asparagine-liked oligosaccharide, a 20% aceticanhydride/2-propanlol:methanol=1:1 solution was used for capping theunreacted amino group of asparagine-linked oligosaccharide, and then theprotective Fmoc group was removed. The resin was washed with isopropanoland DMF and dried. The condensation of Fmoc-asparagine-linkedasialooligosaccharide was conducted in the same manner as thecondensation of benzyl-Fmoc-asparagine-liked disialooligosaccharide ofExample 3.

Shown below is data as to ¹H-NMR (30° C.) of the glycopeptide obtainedwhich is in the form ofHOOC-Ser-Ser-Asn(disialooligo)-Val-Leu-Leu-Ala-Asn(asialooligo)-Val-Leu-Leu-Ala-NH₂.δ 5.22, 5.21 (each s, each 1H, Man4-H1, ManD-H1), 5.11 (d, 2H,GlcNAc1-H1, GlcNAcA-H1), 5.03, 5.01 (each s, each 1H, Man4′-H1,ManD′-H-1), 4.86 (2H, Asnα), 4.69-4.66 (GlcNAc2, B, 5, 5′, E, E′-H1),4.61-4.48 (Leuα×4, Serα×2, Gal6, 6′, F, F′-H1), 4.33 (bs, 2H, Man3,C—H2), 4.28 (bs, 2H, Man4, D-H2), 4.20 (bs, 2H, Man4′, D′-H2), 4.20-4.17(Valα×2, Alaα×2), 3.00 (dd, 2H, Asnβ×2), 2.83 (dd, 2H, Asnβ×2), 2.76(dd, 2H, NeuAc7, 7′-H3eq), 1.82 (dd, 2H, NeuAc7, 7′-H3ax), 2.16-2.10(Ac×10, Valβ), 1.70-1.60 (m, Leuβ, γ), 1.60, 1.49 (each d, each 3H,Alaβ), 1.02-0.96 (m, 36H, Val-CH₃×4, Leu-CH₃×8)

Reference Example 6

The asparagine-linked oligosaccharide represented by Asn(asialooligo)was prepared according to Examples of Japanese Patent Application No.2001-185685. NMR data as to the product is given below.

¹H-NMR (30° C.) δ 5.12 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.7 Hz,GlcNAc1-H-1), 4.92 (s, 1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.62 (d,1H, J=8.0 Hz, GlcNAc2-H-1), 4.58 (d, 2H, J=7.8 Hz, GlcNAc5, 5′-H-1),4.47 (d, 2H, J=7.9 Hz, Gal6, 6′-H-1), 4.24 (bd, 1H, Man3-H-2), 4.19(bdd, 1H, J=3.2 Hz, 1.4 Hz, Man4′-H-2), 4.12 (bdd, 1H, J=3.2 Hz, 1.4 Hz,Man4-H-2), 2.93 (dd, 1H, J=4.5 Hz, 17.0 Hz, Asn-βCH), 2.93 (dd, 1H,J=6.8 Hz, 17.0 Hz, Asn-βCH), 2.08 (s, 3H, Ac), 2.05 (s, 6H, Ac×2), 2.01(s, 3H, Ac)

Also, Fmoc-asparagine-linked asialooligosaccharide was obtained in thesame manner as in Reference Example 2. NMR data thereof is given below.

¹H-NMR (D₂O, 30° C.) δ 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H,m, Fmoc), 5.12 (1H, s, Man4-H1), 5.06 (1H, d, GlcNAc1-H1), 4.93 (1H, s,Man4′-H1), 4.82 (1H, s, Man3-H1), 4.69 (1H, d, GlcNAc2-H1), 4.67 (2H, d,GlcNAc5, 5′-H1), 4.53 (2H, d, Gal6, 6′-H1), 4.34 (1H, d, Man3-H2), 4.27(1H, d, Man4′-H2), 4.19 (1H, d, Man4-H2), 3.03 (1H, bdd, Asn-βCH), 3.00(1H, bdd, Asn-βCH), 2.15 (12H, s×4, -Ac)

Reference Example 7 Preparation of Benzyl3,6-O-pivaloyl-β-D-galactopyranoside (18)

(1) Preparation of Compound (17)

Sodium acetate (5 g, 69 mmols) was dissolved in acetic anhydride (60ml), the solution was heated, and D-galactose (16) (10 g, 55 mmols) wasthereafter added in small portions to the solution. The mixture wasreflux with heating for 2 hours, and the completion of reaction wasthereafter recognized by TLC (toluene:ethyl acetate=5:1). The reactionmixture was returned to room temperature and then poured into 300 cc ofice water. The resulting precipitate was collected by filtration. Theprecipitate was dissolved in ethanol (14 ml) for recrystallization,giving 9.0 g of Compound (17) (41% in yield).

(2) Preparation of Compound (18)

Compound (17) (4.3 g, 11 mmols) was dissolved in methylene chloride (120ml), and the solution was thereafter cooled to −20° C. under an argonstream. Subsequently, tin tetrachloride (3.1 g, 12 mmols) was added tothe solution, the mixture was stirred for 20 minutes, benzyl alcohol(2.3 g, 22 mmols) was then added to the mixture, and the reactiontemperature was returned to room temperature. After the completion ofreaction was recognized by TLC (hexane:ethyl acetate=1:1), the reactionmixture was poured into a saturated aqueous solution of sodiumhydrogencarbonate, followed by extraction with methylene chloride. Themethylene chloride layer was dried over anhydrous magnesium sulfate,then filtered and concentrated in a vacuum. The residue was dried in adesiccator, thereafter dissolved in distilled methanol (80 ml), sodiummethoxide (431 mg, 5.5 mmols) was added to the solution, and the mixturewas stirred under an argon stream. After the completion of reaction wasrecognized by TLC (ethyl acetate:methanol:water=10:5:1), the reactionmixture was neutralized with a cation-exchange resin IR-120(+) toterminate the reaction. The resin was filtered off for removal, and thefiltrate was concentrated in a vacuum. The residue was dried in adesiccator, thereafter dissolved in pyridine (44 ml), and the reactionmixture was cooled to 0° C. Pivaloyl chloride (4.6 g, 38.5 mmols) wasadded to the reaction mixture, and the mixture was returned to roomtemperature and stirred under an argon stream for 1 hour. After thecompletion of reaction was recognized by TLC (hexane:ethyl acetate=2:1),the reaction mixture was cooled to 0° C., and methanol was thereafteradded to the mixture to terminate the reaction. The reaction mixture wasconcentrated as it was in a vacuum, the residue was then dissolved inethyl acetate, the solution was washed with a saturated aqueous solutionof sodium chloride and water, and dried over anhydrous magnesium sulfateto evaporate off the ethyl acetate. After the magnesium sulfate wasremoved by filtration, the filtrate was concentrated in a vacuum. Theresidue was purified by silica gel column chromatography (developingsolvent: hexane:ethyl acetate=2:1), giving Compound (18) (2.8 g, yield58%).

Reference Example 8 Preparation of Benzyl2-O-chloroacetyl-4-deoxy-4-fluoro-3,6-di-O-pivaloyl-β-D-glucopyranoside(20)

(1) Preparation of Benzyl2-O-chloroacetyl-3,6-di-O-pivaloyl-β-D-galactopyranoside (19)

Compound 18 (200 mg, 0.455 mmols) was dissolved in dichloromethane (7.8ml) and pyridine (1.3 ml), chloroacetic anhydride (155 mg, 0.91 mmol)was added to the solution, and the mixture was reacted with stirring at−15° C. under an argon stream for 15 minutes. After the completion ofreaction was recognized, the chloroacetic anhydride was quenched withmethanol (5 ml), and the reaction mixture was azeotropically boiled withtoluene three times for concentration in vacuum. The residue wasextracted with ethyl acetate, and the extract was washed with asaturated aqueous solution of sodium chloride. The organic layer wasdried over anhydrous magnesium sulfate, followed by filtration andconcentration. The residue was purified by silica gel columnchromatography (ethyl acetate:hexane=1:4), giving Compound (19) (in anamount of 172 mg, 73.5% in yield).

¹H-NMR (400 MHz, CDCl₃) δ 7.37-7.29 (m, 5H, Ph), 5.39 (dd, 1H,J_(1, 2)=8.0 Hz, J_(2, 3)=10.4 Hz, H-2), 4.89 (dd, 1H, J_(3, 4)=3.4 Hz,H-3), 4.89, 4.62 (2d, 2H, J=12.5 Hz, OCH ₂Ph), 4.53 (d, 1H, H-1), 4.37(dd, 1H, J_(6a, 6b)=11.5 Hz, J_(6a, 5)=6.0 Hz, H-6a), 4.32 (dd, 1H,J_(6b, 5)=6.6 Hz, H-6b), 4.00 (m, 1H, H-4), 3.92 (s, 2H, COCH ₂Cl), 3.75(dd, 1H, H-5), 1.23, 1.19 [2s, 18H, COC(CH ₃)₃]

¹³C-NMR (400 MHz, CDCl₃) δ 178.33, 177.57, 165.92, (C═O), 136.66,128.48, 128.07, 127.89 (Ph), 99.16 (C-1), 72.82 (C-3), 72.35 (C-5),70.92 (C-2), 70.49 (OCH₂Ph), 67.29 (C-4), 62.30 (C-6), 40.40 (COCH₂Cl),38.95, 38.80 [COC(CH₃)₃], 27.14, 26.98 [COC(CH₃)₃]

¹H-NMR and ¹³C-NMR were measured using Bruker's AVANCE 400 (mentioned as400 MHz). When the solvent was deuteriochloroform, trimethylsilane wasused as internal standard. When other deuteriated solvents were used,the peak of the solvent was used as a reference. Chemical shifts wereindicated by δ (ppm), and the coupling constants by J (Hz). Used forsilica gel chromatography were Merck Silicage 160, 70-230 mesh or230-400 mesh, and spherical silica gel which was Silica Gel 60(Spherical), product of Kanto Chemical Co., Ltd. Used for detectingreactions (for TLC) was DC-Platten Kieselgel 60 F254 (Artl, 05715),product of E. Merk. The columns used for high performance chromatography(HPLC) were COSMOSIL 5C₁₈-AR Packed Column [φ4.6×150 mm], product ofNakaraitesuku Co., Ltd. The spectrophotofluorometer used was FP-210Spectrofluorometer, product of JASCO.

(2) Preparation of Benzyl2-O-chloroacetyl-4-deoxy-4-fluoro-3,6-di-O-pivaloyl-β-D-glucopyranoside(20)

Compound (19) (300 mg, 0.583 mmol) was dissolved in dichloromethane (5.8ml), and diethylaminosulfatrifluoride (DAST) was added to the solutionwith stirring under an argon stream at −15° C. The mixture was returnedto room temperature 10 minutes after the addition of DAST and reactedfor 1 hour. Disappearance of the material was confirmed by TLC, the DASTwas quenched with methanol (3 ml), and the reaction mixture wasconcentrated in a vacuum. The residue was purified by silica gelchromatography (ethyl acetate:hexane=1:6), giving Compound (20) (in anamount of 211 mg, yield 70%).

¹H-NMR (400 MHz, CDCl₃) δ 7.37-7.27 (m, 5H, Ph), 5.31 (ddd, 1H,J_(3, F)=14.3 Hz, J_(3, 4)=9.69 Hz, J_(2, 3)=9.63 Hz, H-3), 5.04 (dd,1H, J_(1, 2)=7.93 Hz, H-2), 4.86 (d, 1H, J=12.2 Hz, OCH ₂Ph), 4.60 (d,1H, H-1), 4.59 (d, 1H, OCH ₂Ph), 4.44 (ddd, 1H, J_(4, 5)=9.04 Hz,J_(4, F)=50.6 Hz, H-4), 4.43 (ddd, 1H, J_(6a, 6b)=12.1 Hz,J_(6a, 5)=2.41 Hz, J_(6a, F)=2.23 Hz, H-6a), 4.24 (ddd, 1H,J_(6b, 5)=5.67 Hz, J_(6b, F)=1.28 Hz, H-6b), 3.93 (s, 2H, OCOCH ₂Cl),3.75 (m, 1H, H-5), 1.25, 1.18 [2s, 18H, OCOC(CH₃)₃]

¹³C-NMR (400 MHz, CDCl₃) δ 177.94, 117.43, 165.88 (C═O), 136.34, 128.55,138.23, 127.92 (Ph), 98.68 (C-1), 87.35 (d, J_(4, F)=188.62 Hz, C-4),72.65 (d, J_(2, F)=7.96 Hz, C-2), 72.05 (d, J_(3, F)=20.02 Hz, C-3),71.49 (d, J_(5, F)=23.09 Hz, C-5), 70.80 (OCH₂Ph), 62.12 (C-6), 40.30(OCOCH₂Cl), 38.87 [OCOC(CH₃)₃], 27.17, 26.92 [OCOC(CH₃)₃]

Reference Example 9 Preparation of Benzyl2-azido-2,4-dideoxy-4-fluoro-3,6-di-O-pivaloyl-β-D-mannopyranoside (22)

(1) Preparation of Benzyl4-deoxy-4-fluoro-3,6-di-O-pivaloyl-β-D-glucopyranoside (21)

Compound (20) (625 mg, 1.21 mmols) was dissolved in methanol (24.2 ml),and sodium methoxide (13.1 mg, 0.6 mmol) was added to the solution withstirring under an argon stream at −15° C. Disappearance of the materialwas confirmed by TLC 30 minutes later, and the reaction mixture wasneutralized (pH 6-7) with a cation-exchange resin IR-120(+). After theresin was filtered off, the filtrate was concentrated in a vacuum. Theresidue was purified by silica gel chromatography (ethylacetate:hexane=1:4), giving Compound (21) (in an amount of 395 mg, yield74%).

¹H-NMR (400 MHz, CDCl₃) δ 7.38-7.29 (m, 5H, Ph), 5.18 (ddd, 1H,J_(3, F)=14.8 Hz, J_(3, 4)=9.51 Hz, J_(2, 3)=8.99 Hz, H-3), 4.90 (d, 1H,J=11.7, OCH ₂Ph), 4.63 (d, 1H, OCH ₂Ph), 4.47 (ddd, 1H, J_(5, 6a)=2.43Hz, J_(6a, F)=2.2 Hz, H-6a), 4.47 (d, 1H, J_(1, 2)=7.7 Hz, H-1), 4.38(ddd, 1H, J_(4, 5)=8.96 Hz, J_(3, 4)=9.67 Hz, J_(4, F)=50.8 Hz, H-4),4.23 (ddd, 1H, J_(6a, 6b)=12.0 Hz, J_(6b, 5)=6.05 Hz, J_(6b, F)=1.26 Hz,H-6b), 3.75 (m, 1H, H-5), 3.54 (m, 1H, J_(2, OH)=2.70 Hz, H-2), 1.27,1.26 [2s, 18H, OCOC(CH₃)₃]

¹³C-NMR (400 MHz, CDCl₃) δ 178.17, 177.94 (C═O), 136.54, 128.54, 128.17,128.12 (Ph), 101.31 (C-1), 87.45 (d, J_(4, F)=187.39 Hz, C-4), 74.17 (d,J_(3, F)=18.88 Hz, C-3), 72.45 (d, J_(2, F)=7.56 Hz, C-2), 71.45 (d,J_(5, F)=23.26 Hz, C-5), 71.09 (OCH₂Ph), 62.44 (C-6), 38.90, 38.85[OCOC(CH₃)₃], 27.14, 26.99 [OCOC(CH₃)₃]

(2) Preparation of Benzyl2-azido-2,4-dideoxy-4-fluoro-3,6-di-O-pivaloyl-β-D-mannopyranoside (22)

To a solution of pyridine (22.2 μl, 0.274 mmol) in dichloromethane (370μl) was added dropwise trifluoromethane-sulfonic anhydride (46 μl, 0.274mmol) at 0° C., and 15 minutes later, a solution of Compound (21) indichloromethane (1 ml) was added dropwise to the mixture at 0° C.Disappearance of the material was confirmed by TLC, and the reactionmixture was diluted with dichloromethane. The organic layer was washedwith a saturated aqueous solution of sodium hydrogencarbonate, saturatedsodium chloride aqueous solution and water, dried over anhydrousmagnesium sulfate and thereafter concentrated. The residue was furtherdried by a vacuum pump, and then dissolved in benzene (1 ml). Sodiumazide (13 mg, 0.206 mmol) and tetraammonium chloride (57 mg, 0.206 mmol)were added to the solution under an argon stream at room temperature,and the mixture was reacted at 40° C. The disappearance of the materialwas confirmed by TLC, and the reaction mixture was thereafterconcentrated in a vacuum. The residue was subjected to extraction withethyl acetate, and the extract was washed with a saturated sodiumchloride aqueous solution and water, dried over anhydrous magnesiumsulfate and thereafter concentrated. The residue was purified by silicagel column chromatography (ethyl acetatae:hexane=1:4), affordingCompound (22) (in an amount of 30.4 mg, 95% in yield).

¹H-NMR (400 MHz, CDCl₃) δ 7.39-7.32 (m, 5H, Ph), 4.99 (ddd, 1H,J_(3, F)=13.18 Hz, J_(3, 4)=9.27 Hz, J_(2, 3)=3.87 Hz, H-3), 4.93 (d,1H, J=12.07 Hz, OCH ₂Ph), 4.67 (d, 1H, J_(1, 2)=1.18 Hz, H-1), 4.63 (d,1H, OCH ₂Ph), 4.51 (ddd, 1H, J_(6a, 6b)=11.95 Hz, J_(6a, 5)=2.54 Hz,J_(6a, F)=2.08 Hz, H-6a), 4.23 (ddd, 1H, J_(6b, 5)=6.14 Hz,J_(6b, F)=1.14 Hz, H-6b), 4.08 (m, 1H, H-2), 3.64 (m, 1H, H-5), 1.26[2s, 18H, OCOC(CH ₃)₃]

¹³C-NMR (400 MHz, CDCl₃) δ 178.01, 177.68 (C═O), 136.06, 128.63, 128.31,128.14 (Ph), 97.25 (C-1), 85.51 (d, J_(4, F)=183.97, C-4), 72.01 (d,J_(5, F)=23.89, C-5), 71.73 (d, J_(3, F)=18.98, C-3) 70.57 (OCH₂Ph),62.42 (C-2, C-6), 39.08, 38.90 [OCOC(CH₃)₃], 27.18, 26.95 [OCOC(CH₃)₃]

Reference Example 10 Preparation ofN-Acetyl-4-deoxy-4-fluoro-D-mannosamine 24

(1) Preparation of Benzyl2-azido-2,4-dideoxy-4-fluoro-β-D-mannopyranoside (23)

Compound (22) (180 mg, 0.387 mmol) was dissolved in methanol (8 ml),sodium methoxide (922 mg, 9.67 mmols) was added to the solution, and themixture was reacted with stirring at 40° C. TLC revealed 4.5 hours laterthat the reaction mixture collected into a spot, and the mixture wasneutralized with a cation-exchange resin IR-120(+), followed byfiltration and concentration. The residue was purified by silica gelcolumn chromatography (ethyl acetate:hexane=1:1), giving Compound (23)(in an amount of 105.3 mg, 91.6% in yield).

¹H-NMR (400 MHz, CDCl₃) δ 7.40-7.31 (m, 5H, Ph), 4.96 (d, 1H, J=12.13Hz, OCH ₂Ph), 4.71 (d, 1H, J_(1, 2)=1.33 Hz, H-1), 4.69 (d, 1H, OCH₂Ph), 4.49 (ddd, 1H, J_(4, F)=51.06 Hz, J_(4, 5)=9.19 Hz, J_(3, 4)=9.20Hz, H-4), 4.02 (m, 1H, H-2), 3.93 (dddd, 1H, J_(6a, 6b)=12.19 Hz,J_(6a, 5)=2.31 Hz, J_(6a, F)=2.32 Hz, J_(6a, OH)=6.20 Hz, H-6a),3.89-3.77 (m, 2H, H-3, H-6b), 3.39 (m, 1H, H-5)

¹³C-NMR (400 MHz, CDCl₃) δ 136.39, 128.62, 128.24, 127.83 (Ph), 98.63(C-1), 88.19 (d, J_(4, F)=178.91 Hz, C-4), 73.95 (d, J_(5, F)=25.48 Hz,C-5), 71.18 (OCH₂Ph), 71.16 (d, J_(3, F)=19.69 Hz, C-3), 64.48 (d,J_(2, F)=8.42 Hz, C-2), 61.39 (C-6)

(2) Preparation of N-Acetyl-4-deoxy-4-fluoro-D-mannosamine (24)

Compound (23) (105 mg, 0.353 mmol) was dissolved in methanol (7 ml),acetic anhydride (333 μl, 3.53 mols) was added to the solution, acatalytic amount of 10% Pd/C was thereafter added to the mixture, andthe resulting mixture was stirred at room temperature after replacingthe atmosphere in the reactor with hydrogen. TLC indicated disappearanceof the material 2 hours later, followed by filtration with activatedcarbon and concentration. The residue was purified by silica gel columnchromatography (ethyl acetate:methanol=5:1), giving Compound (24) (in anamount of 57 mg, 72% in yield).

¹H-NMR (400 MHz, D₂O) δ 5.23 (dd, 1H, J_(1, 2)=2.69 Hz, J_(1, F)=1.44Hz, H-1-α), 4.65 (ddd, 1H, J_(4, F)=50.94 Hz, J_(3, 4)=9.06 Hz,J_(4, 5)=9.58 Hz, H-4-α), 4.47 (m, 1H, H-2-α), 4.43 (ddd, 1H,J_(3, F)=14.28 Hz, J_(2, 3)=4.9 Hz, H-3-α), 4.16 (m, 1H, H-5-α), 3.95(m, 2H, H-6a-α, H-6b-α), 2.14 (s, 3H, NHCOCH ₃-α)

¹³C-NMR (400 MHz, D₂O) δ 175.27 (C═O-α), 93.46 (C-1-α), 88.30 (d,J_(4, F)=177.00 Hz, C-4-α), 69.91 (d, J_(5, F)=24.41 Hz, C-5-α), 67.60(d, J_(3, F)=18.74 Hz, C-3-α), 60.36 (C-6), 54.12 (d, J_(2, F)=8.68 Hz,C-2-α), 22.31 (NHCOCH₃-α)

Reference Example 11 Preparation of5-Acetamido-3,5,7-trideoxy-7-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (25)

Compound (24) (50 mg, 0.224 mmol), sodium piruvate (123 mg, 1.12 mmols)and bovine serum albumin (5 mg) were dissolved in a sodium phosphatebuffer solution (100 mM, pH 7.5, 3.4 ml), and aldolase sialate wasthereafter added to the solution to start a reaction at roomtemperature. The reaction mixture was freeze-dried 24 hours later. Theproduct was dissolved in a small amount of water and applied to ananion-exchange resin column (AG 1-X8, 200-400 mesh, formate form). Afterpassing 300 ml of water through the column, the desired product waseluted with 1M formic acid, and the eluate was concentrated in a vacuum.The residue was purified by a gel filtration column (Sephadex G-15,water), giving Compound (25) (in an amount of 40 mg, 58.9% in yield).

¹H-NMR (400 MHz, D₂O) δ 4.61 (dd, 1H, J_(7, 8)=8.97 Hz, J_(7, F)=45.56Hz, H-7), 4.18 (dd, 1H, J_(5, 6)=10.63 Hz, J_(6, F)=29.86 Hz, H-6), 4.15(m, 1H, H-4), 4.07 (m, 1H, H-8), 4.02 (dd, 1H, J_(4, 5)=100.10 Hz, H-5),3.90 (ddd, 1H, J_(9a9b)=12.18 Hz, J_(9a, 8)=2.77 Hz, J_(9a, F)=2.86 Hz,H-9a), 3.76 (ddd, 1H, J_(9b, 8)=5.33 Hz, J_(9b, F)=2.06 Hz, H-9b), 2.40(dd, 1H, J_(3eq, 3ax)=13.00, J_(3eq, 4)=4.88 Hz, H-3 eq), 2.15 (s, 3H,OCOCH ₃), 2.00 (dd, 1H, J_(3ax, 4)=11.70 Hz, H-3ax)

¹³C-NMR (400 MHz, D₂O) δ 175.17, 173.68 (C═O), 96.01 (C-1), 89.12 (d,J_(7, F)=179.23 Hz, C-7), 69.67 (d, J_(6, F)=17.41 Hz, C-6), 68.31 (d,J_(8, F)=26.50 Hz, C-8), 67.26 (C-4), 62.70 (C-6), 52.17 (C-5), 39.19(C-3), 22.61 (NHCOCH₃

Reference Example 12 Preparation of5-Acetamido-3,5,8-trideoxy-8-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (27)

5-Acetamido-3,5,8-trideoxy-8-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (27) was prepared from Sialic acid (26) according to the schemegiven below.

(a) (1) Dowex 50-X8, dist. MeOH, (2) Acetone dimethyl acetal, Camphorsulfonic acid, MeCN, y=73%;(b) (1) BaO, Ba(OH)₂, BnBr, DMF, (2) CH₂N₂, (3) 60% AcOH, y=61.8%;(c) (1) Dibutyltin oxide, toluene:MeOH=5:1, (2) tetra-n-butyl ammoniumbromide, BnBr, toluene, y=74.3%;(d) (1) DMSO, Oxalyl chloride, TEA, CH₂Cl₂, (2) BH₃NH₃, MeOH, y=73.2%;(e) DAST, CH₂Cl₂, y=29.8%;(f) Pd/C, AcOH, y=74.2%;(g) (1) 0.3N NaOH, (2) Amberlyst 15H(+), 0.016N HCl, y=72.6%(a) (1) Sialic acid (26) (1.02 g, 3.31 mmols) was dissolved in distilledmethanol (150 ml), a cation-exchange resin, DOWex 50W-X8, (2.0 g) wasadded to the solution and the mixture was refluxed with heating for 24hours for reaction. The end point of the reaction was confirmed bysubjecting a portion of the reaction mixture to NMR spectroscopy. Thereaction mixture was filtered, and methanol (100 ml) was added again tothe resin, followed by stirring for 1 hour to collect the compoundadsorbed by the resin. The resulting solution was filtered against, andthe filtrate was combined with the filtrate obtained first, and thecombined filtrate was concentrated in a vacuum to obtain a compound.

(2) The compound (5.05 g, 14.97 mmols) obtained above was dissolved indistilled acetone, and camphorsulfonic acid (498 mg, 2.14 mmols) wasadded to the solution with stirring under an argon stream at roomtemperature. Acetone dimethyl acetal (2.75 ml, 22.36 mmols) wasthereafter added dropwise in small portions to the mixture to effect areaction for 30 minutes. After the completion of reaction was confirmed,the reaction was terminated by the addition of triethylamine (2 ml), andthe reaction mixture was concentrated in a vacuum. The residue waspurified by silica gel column chromatography (ethylacetate:methanol=20:1) to obtain acetonide derivative (α:β=1:10, yield5.29 g).

(b) (1) The acetonide derivative (3.2 g, 8.48 mmols) obtained above wasdissolved in N,N-dimethylformamide (43 ml), and barium oxide (9.3 g,60.65 mmols) and barium hydroxide octahydrate (2.4 g, 7.61 mmols) wereadded to the solution. Subsequently, the mixture was stirred at roomtemperature, with benzyl bromide (10 ml, 84.1 mmols) added thereto.After the disappearance of the material was confirmed by TLC, thereaction mixture was diluted with dichloromethane and washed with a 1%formic acid aqueous solution and water, and the organic layer was driedover anhydrous magnesium sulfate. The magnesium sulfate was filteredoff, and the organic layer was concentrated in a vacuum.

(2) The residue was dissolved in a solvent mixture of ethanol (25 ml)and benzene (50 ml), and a solution of diazomethane (42.5 mmols) inether was added to the solution. The diazomethane used was produced byadding p-toluenesulfonyl-N-nitrous amide to a mixture solution of etherand ethanol, and adding a 50% & potassium hydroxide dropwise to themixture. After the addition of the diazomethane, the mixture was reactedat room temperature for 10 minutes. After the disappearance of thematerial was confirmed by TLC, an excess of diazomethane was quenchedwith acetic acid (12 ml), followed directly by concentration in avacuum.

(3) Subsequently, the residue was dissolved in a 60% aqueous solution ofacetic acid, followed by a reaction at 60° C. for 12 hours. After thedisappearance of the material was confirmed by TLC, the reaction mixturewas concentrated in a vacuum. The residue was purified by silica gelcolumn chromatography (ethyl acetate:methanol=15:1) to obtain a compound(α:β=1:24, yield 2.7 g).

(c) (1) The compound (1.08 g, 2.08 mmols) was dissolved in toluene (30ml) and methanol (6.5 ml), dibutyl tin oxide (780 mg, 3.48 mmols) wasadded to the solution, and the mixture was reacted at 85° C. for 2hours. The reaction mixture was thereafter concentrated in a vacuum, andthe residue was azeotropically boiled with thoroughly dehydrated toluenethree times.

(2) The residue was dissolved in toluene (24 ml) again,tetrabutylammonium bromide (1.00 g, 3.48 mmols) and benzyl bromide (977ml, 10.4 mmols) were added to the solution, and the mixture was reactedat 80° C. for four hours. After the disappearance of the material wasconfirmed by TLC, the reaction mixture was returned to room temperatureand concentrated in a vacuum. The residue was purified by silica gelcolumn chromatography (ethyl acetate:hexane=4:1) to obtain a4,7,9-benzyl compound (α:β=1:10, yield 1.15 g).

(d) (1) Oxalyl chloride (1.82 g, 14.3 mmols) was added todichloromethane (13 ml), and the mixture was cooled to −78° C. A mixturesolution of dimethyl sulfoxide (1.3 ml, 17.9 mmols) and dichloromethane(5 ml) was added to the mixture 15 minutes later, followed by stirringat −78° C. again. A solution of the 4,7,9-benzyl compound (2.18 mg, 3.59mmols) obtained above in dichloromethane (18 ml) was slowly added to theresulting mixture 20 minutes later. The mixture was stirred at −78° C.for 20 minutes, triethylamine (4.00 ml, 28.7 mmols) was thereafter addedto the mixture, followed by stirring for 10 minutes, and the reactiontemperature was returned to room temperature. The appearance of thematerial was confirmed by TLC, the reaction mixture was then dilutedwith dichloromethane and washed with an aqueous solution of sodiumhydrogencarbonate and saturated solution of sodium chloride, and theorganic layer was dried over anhydrous magnesium sulfate. After themagnesium sulfate was removed by filtration, the organic layer wasconcentrated in a vacuum.

(2) The residue was directly dissolved in methanol (16 ml) withoutpurification, the solution was cooled to −15° C., BH₃NH₃ (122 mg, 3.95mmols) were added to the solution, and the reaction temperature wasreturned to room temperature. The disappearance of the material wasconfirmed by TLC, and the reaction mixture was thereafter concentratedas it was in a vacuum. The residue was purified by silica gel columnchromatography (ethyl acetate:hexane=2:1), affording an 8-epimer (yield1.05 g).

(e) The 8-epimer (533 mg, 0.87 mmols) obtained above was dissolved indichloromethane (13 ml), followed by cooling to −15° C. under an argonstream. Dimethylaminosulfur trifluoride (580 ml, 3.51 mmols) was slowlyadded to the solution, followed by stirring for 30 minutes, and thereaction temperature was raised to 40° C., further followed by stirringfor 16 hours. After the reaction was terminated by the addition ofmethanol, the reaction mixture was concentrated in a vacuum. The residuewas purified by silica gel column chromatography (ethylacetate:hexane=2:3), affording an 8-fluoro compound (yield 144 mg).(f) The 8-fluoro compound (120 mg, 0.197 mmols) obtained above wasdissolve in acetic acid (4 ml), 10% Pd/C (120 mg) was added to thesolution under an argon stream, the atmosphere was replaced by hydrogen,and the mixture was thereafter stirred at room temperature. Thecompletion of reaction was confirmed by TLC 2 hours later, the reactionmixture was filtered with activated carbon, and the filtrate wasconcentrated in a vacuum. The residue was purified by silica gel columnchromatography (ethyl acetate:methanol=6:1) to obtain a compound (yield57 mg).(g) (1) The compound (50 mg, 0.147 mmol) obtained above was dissolved inmethanol (2 ml), 0.3 N sodium hydroxide aqueous solution (2 ml) wasadded to the solution, and the mixture was stirred at room temperature.The completion of reaction was confirmed by TLC, and the reactionmixture was thereafter neutralized with IR-120(+). IR-120(+) was removedby filtration, and the filtrate was concentrated in a vacuum.

(2) The residue was dissolved as it was in 0.016 N hydrochloric aqueoussolution (5 ml), Amberlyst 15(H+) (150 mg) was added to the solution,and the mixture was reacted at 75° C. for 24 hours. The completion ofreaction was confirmed by NMR spectroscopy, and the reaction mixture wasconcentrated in a vacuum. The residue was placed on AG1xX8 (200-400mesh, formate form), 150 ml of water was passed through the column, anda 1M formic acid aqueous solution was thereafter applied for elution,giving 8-fluorosialic acid (27) (yield 33 mg).

NMR data as to 8-fluorosialic acid is given below.

¹H-NMR (400 MHz, D₂O) δ 4.69 (dddd, 1H, J_(8,F)=48.7 Hz, J_(8,9a)=5.0Hz, J_(8, 9b)=3.5 Hz, H-8), 4.03 (ddd, 1H, J_(4,5)=10.0 Hz,J_(3ax,4)=11.1 Hz, J_(3eq, 4)=4.7 Hz, H-4), 3.95 (dd, 1H, J_(4,5)=10.0Hz, J_(5,6)=9.9 Hz, H-5), 3.94 (ddd, 1H, J_(6, 7)=˜0 Hz, J_(7,8)=6.8 Hz,J_(7,F)=14.0 Hz, H-7), 3.88 (ddd, 1H, J_(9a9b)=13.3 Hz, J_(9a,8)=3.5 Hz,J_(9b,F)=28.0 Hz, H-9b), 3.86 (dd, 1H, J_(5,6)=9.9 Hz, J_(6,7)=˜0 Hz,H-6), 3.72 (ddd, 1H, J_(9a,9b)=5.33 Hz, J_(9a,8)=5.0 Hz, J_(9a,F)=30.6Hz, H-9a), 2.28 (dd, 1H, J_(3eq,3ax)=13.00, J_(3eq,4)=4.6 Hz, H-3 eq),2.05 (s, 3H, Ac), 1.87 (dd, 1H, J_(3ax,4)=11.1 Hz, J_(3eq,3ax)=13.00,H-3ax)

Reference Example 13 Preparation of5-Acetamido-3,5,9-trideoxy-9-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (28)

5-Acetamido-3,5,9-trideoxy-9-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (28) was prepared from Sialic acid (26) according to the schemegiven below.

(a) (1) Dowex 50W-X8, MeOH, reflux, (2) TrCl, pyridine, 72%

(b) (1) BaO, Ba(OH), (2) DMF, (3) CH₂CN₂, 88% (c) AcOH, 100° C., 78%

(d) (1) Tf₂O, pyridine, CH₂Cl₂, (2) TASF, CH₂Cl₂, 52%

(e) H₂, Pd/C(10%), AcOH, 86% (f) NaOHaq. (g) 0.02N HClaq., Amberlyst15(H+), 86%

The above reactions were conducted according to the literature below.

T. Miyazaki, T. Sakakibara, H. Sato, Y. Kajihara; ChemoenzymaticSynthesis of the 9-Deoxy-9-fluoro-[3-13C]-NeuAc-a-(2,6)-[U-13C]-Gal-b-Sequence on An Intact Glycoprotein. J. Am. Chem. Soc., 121, 1411-1412(1999).

Reference Example 14 Preparation of CMP-7-fluorosialic acid Derivative

(a) (1) Dowex 50-X8, MeOH, (2) Ac₂O, 60% HClO₄;

(b) (1) 1H-Tetrazole, CH₃CN, (2) t-BuOOH, CH₃CN, (3) DBU, CH₃CN, (4)NaOMe, MeOH, H₂O

A 0.074 mmol quantity of Compound (25), which is a fluorosialic acidderivative, was dissolved in distilled methanol (3 ml), Dowex-50W-X8 (65mg) was added to the solution with stirring under an argon stream, andthe mixture was reacted for 3 hours. After the completion of reactionwas recognized, the reaction mixture was filtered and concentrated in avacuum. The residue was dissolved in acetic anhydride (200 μl), asolution (22 μl) of acetic anhydride and 60% perchloric acid (15:1) wasadded to the solution with stirring at −20° C., and the mixture wasreacted at 0° C. for 40 minutes. After the completion of reaction wasrecognized, the reaction mixture was diluted with ethyl acetate andwashed with a saturated aqueous solution of sodium hydrogencarbonate.The organic layer was dried over anhydrous magnesium sulfate, followedby filtration and subsequent concentration in a vacuum to obtain aresidue containing Compound (29). The residue and CMP-5′-phosphoamiditederivative (30) (136 mg, 0.23 mmol) were azeotropically boiled withrespective portions of benzene three times, the residue was dissolved indistilled acetonitrile (100 μl) each time, and the resulting solutionswere mixed together. To the resulting solution was added 1H-tetrazole(17 mg, 0.23 mmol) with stirring in ice water under an argon stream. Themixture was returned to room temperature 5 minutes later, followed by afurther reaction for 10 minutes. After the completion of reaction wasrecognized, the reaction mixture was diluted with ethyl acetate andwashed with a saturated aqueous solution of sodium hydrogencarbonate andsaturated aqueous solution of sodium chloride. The organic layer wasdried over anhydrous magnesium sulfate, followed by filtration andconcentration at a temperature of up to 30° C. and further by azeotropicboiling with toluene twice to remove water. Distilled acetonitrile (400μl) was added to the residue, and 2.5M t-BuOOH toluene solution (290 μl)was added dropwise to the mixture with ice cooling under an argonstream. The mixture was returned to room temperature 5 minutes later,followed by stirring for 20 minutes. After completion of reaction wasrecognized, dimethyl sulfide (53 μl) was added dropwise to the mixture,and the t-BuOOH was quenched. DBU (18 μl) was thereafter added dropwiseto the mixture, followed by stirring at room temperature for 20 minutes.After the completion of reaction was recognized, methanol (0.67 ml),water (1.35 ml) and sodium methoxide (360 mg) were added to the reactionmixture, followed by reaction at room temperature for 16 hours. Afterthe completion of reaction was recognized, the reaction mixture wassubjected to extraction with water, and the extract was washed withdichloromethane. The aqueous layer was concentrated in a vacuum to about8 ml at a temperature of up to 25° C. The resulting aqueous solution waspurified by gel column chromatography (developing solvent: 20 mM ammoniawater, flow rate: 0.3 ml/min), giving CMP-fluoro-sialic acid derivative(31).

The NMR data of CMP-7″-deoxy-7″-fluoro-sialic acid (31) is given below.

¹H-NMR (400 MHz, 50 mM ND₄DCO₃ in D₂O), δ 8.04 (d, 1H, J_(5, 6)=7.6 Hz,H-6), 6.20 (d, 1H, J_(6, 5)=7.6 Hz, H-5), 6.06 (d, 1H, J_(1′, 2′)=4.5Hz, H-1′), 4.54 (dd, 1H, J_(7″, 8″)=9.5 Hz, J_(7″, F)=45.9 Hz, H-7″),4.42˜4.20 (m, 7H, H-2′, H-3′, H-4′, H-5′ a, H-5′ b, H-6″, H-8″), 4.16(ddd, 1H, J_(4″, 3″ eq)=4.7 Hz, J_(4″, 3″ ax)=11.3 Hz, J_(4, 5)=10.3 Hz,H-4″), 4.03 (dd, 1H, J_(5″, 4″)=J_(5″, 6″)=10.3 Hz, H-5″), 3.91 (ddd,1H, J_(9″ a, 9″ b)=12.2 Hz, J_(9″ a, 8″)=2.8 Hz, J_(9″ a, F)=2.8 Hz,H-9″ a), 3.75 (ddd, 1H, J_(9″ a, 9″ b)=12.2 Hz, J_(9″ b, 8)=5.4 Hz,J_(9″ b, F)=2.1 Hz, H-9″ b), 2.61 (dd, 1H, J_(3″eq, 4″)=4.7 Hz,J_(gem)=13.3 Hz, H-3″ eq), 2.14 (s, 3H, Ac), 1.76 (ddd, 1H,J_(3″ ax, 4″)=11.5 Hz, J_(gem)=13.3 Hz, J_(3″ ax, P)=5.6 Hz, H-3″ ax)

Reference Example 15 Preparation of CMP-8″-deoxy-8″-fluoro-sialic acid

CMP-8″-deoxy-8″-fluoro-sialic acid was prepared in the same manner as inReference Example 14 with the exception of using Compound (27) in placeof Compound (25). NMR data is given below.

¹H-NMR (400 MHz, 50 mM ND₄DCO₃ in D₂O) δ 8.08 (d, 1H, J_(5, 6)=7.6 Hz,H-6), 6.20 (d, 1H, J_(6, 5)=7.6 Hz, H-5), 6.09 (d, 1H, J_(1′, 2′)=4.1Hz, H-1′), 4.90 (m, 1H, H-8″), 4.42 (dd, 1H, J_(3′, 2′)=J_(3′, 4′)=4.9Hz, H-3′), 4.39 (dd, 1H, J_(2′, 1′)=4.1 Hz, J_(2′, 3′)=4.9 Hz, H-2′),4.31-4.28 (m, 3H, H-4′, H-5′ a, H-5′ b), 4.15 (ddd, 1H,J_(4″, 3″ eq)=4.4 Hz, J_(4″, 3″ ax)=11.5 Hz, J_(4, 5)=10.5 Hz, H-4″),4.10-3.90 (m, 5H, H-5″, H-6″, H-7″, H-9″ a, H-9″ b), 2.60 (dd, 1H,J_(3″ eq, 4″)=4.4 Hz, J_(gem)=13.1 Hz, H-3″ eq), 2.13 (s, 3H, Ac), 1.77(ddd, 1H, J_(3″ ax, 4″)=11.5 Hz, J_(gem)=13.1 Hz, J_(3″ ax, P)=4.5 Hz,H-3″ ax)

Reference Example 16 Preparation of CMP-9″-deoxy-9″-fluoro-sialic acid

CMP-9″-deoxy-9-fluoro-sialic acid was prepared in the same manner as inReference Example 14 with the exception of using Compound (28) in placeof Compound (25).

Example 5 Preparation of HOOC-Ser-Ser-Asn(Asialooligo)-Val-Leu-Leu-Ala-NH-Dansyl

Into a solid-phase synthesis column was placed 370 mg of HMPA-PEGAresin, which was thoroughly washed with CH₂Cl₂ and DMF.

Fmoc-Ser (OtBu)-OH, 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT)and N-methylimidazole were dissolved in CH₂Cl₂, and the solution wasstirred for 5 minutes and thereafter placed into the solid-phasesynthesis column containing the resin, followed by stirring at roomtemperature for 3 hours. The resin was thereafter washed with methylenechloride, isopropanol and DMF and dried. The unreacted hydroxyl on thesolid phase was thereafter acetylated using a 20% DMF solution of aceticanhydride for 20 minutes for capping. The resin was washed with DMF andstirred along with a 20% piperidine/DMF solution for 20 minutes toremove the protective Fmoc group, whereby resin-Ser-NH₂ was obtained.The product was washed with DMF and dried.

Next, Fmoc-Ser(OtBu)-OH was used with HOBt.H₂O and DIPCDI forcondensation.

Subsequently, Fmoc-asparagine-linked asialooligosaccharide (15) wasdissolved in a 1:1 solvent mixture of DMSO and DMF, and the solution,HATU and DIPEA were stirred at room temperature for 24 hours forcondensation. The resulting resin was washed with DMF and thereafterstirred along with 10% acetic anhydride/2-propanol:methanol for 20minutes for capping. The resin was washed with 2-propanol and DMF, andthereafter stirred along with 20% piperidine/DMF for 20 minutes toremove the protective Fmoc group. The resin was washed with DMF.

The resulting resin, and valine (Val), leucine (Leu), leucine (Leu) andalanine (Ala) were similarly subjected to condensation, followed byremoval of the protective Fmoc group to obtainresin-Ser-Ser-Asn(asialooligo)-Val-Leu-Leu-Ala-NH₂.

Used as the amino acids of valine (Val), leucine (Leu), and alanine(Ala) were each Fmoc-AA-Opfp (AA=amino acid) wherein the carboxyl waspfp-esterified, and 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (Dhbt) wasused for condensation. All condensation reactions were conducted in aDMF solution. For fluorescence marking, the resin was reacted withdansyl chloride and diisopropylethylamine in DMF for 30 minutes. Afterthe completion of dansylation, the resin was washed with DMF and CH₂Cl₂.

To the washed resin was added a 95% aqueous solution of TFA, followed bystirring at room temperature for 3 hours to cut off the resin. The resinwas filtered off. The reaction mixture was concentrated in a vacuum atroom temperature, thereafter dissolved in water and freeze-dried. Theresulting product was purified by HPLC to obtain the desired product,i.e., HOOC-Ser-Ser-Asn(asialooligo)-Val-Leu-Leu-Ala-NH-Dansyl.

(YMC-Pack A-314 S-5 ODS 300×6.0 mm, developing solvents A: 0.1% TFAaqueous solution, B: 0.1% TFA acetonitrile:water=90:10, gradient A 100%0.60 ml/min→B 100% 0.60 ml/min 60 min).

Example 6 Preparation ofHOOC-Ser-Ser-Asn(asialooligo)-Val-Leu-Leu-Ala-NH₂

HOOC-Ser-Ser-Asn (asialooligo)-Val-Leu-Leu-Ala-NH₂ was prepared in thesame manner as in Example 5 with the exception of not conductingdansylation for fluorescence marking.

Example 7

The sialic acid derivative of Reference Example 14 was transferred tothe dansylated asialooligosaccharide peptide obtained in Example 5,using a sialic acid transferase.

Used as the sialic acid transferase was a commercial product derivedfrom a rat recombinant and serving as an α2,3-transferase.

The enzymatic reaction was conducted using the CMP-sialic acidderivative in four equivalents of the dansylated asialooligosaccharidepeptide, 50 mM cacodylic acid buffer (pH 5.0) serving as a reactionsolvent, and a phosphoric acid hydrolase and bovine serum albumin asadded to the solution to be reacted.

The reaction mixture obtained on completion of reaction was freeze-driedas it was. The dried product was purified by HPLC, giving a glycopeptidehaving a dansylated di-7-sialo derivative attached thereto, as givenbelow.

(YMC-Pack A-314 S-5 ODS 300×6.0 mm, developing solvents A: 0.1% TFAaqueous solution, B: 0.1% TFA acetonitrile:water=90:10, gradient A 100%0.60 ml/min→B 100% 0.60 ml/min 60 min)

Example 8

A glycopeptide having a dansylated di-7-sialo derivative as 2,6-linkedthereto, shown below, was obtained in the same manner as in Example 7with the exception of using as the sialic acid transferase a commercialproduct derived from rat liver and serving as an α2,6-transferase andusing a cacodylic buffer adjusted to a pH of 6.0.

Example 9

A glycopeptide having a di-8-sialo derivative as 2,3-linked thereto,shown below, was obtained in the same manner as in Example 7 with theexception of using the sialic acid derivative of Reference Example 15instead of the sialic acid derivative of Reference Example 14.

Example 10

A glycopeptide having a di-8-sialo derivative as 2,6-linked thereto,shown below, was obtained in the same manner as in Example 8 with theexception of using the sialic acid derivative of Reference Example 15instead of the sialic acid derivative of Reference Example 14.

Example 11

A glycopeptide having a di-9-sialo derivative as 2,3-linked thereto,shown below, was obtained in the same manner as in Example 7 with theexception of using the sialic acid derivative of Reference Example 16instead of the sialic acid derivative of Reference Example 14.

Example 12

A glycopeptide having a di-9-sialo derivative as 2,6-linked thereto,shown below, was obtained in the same manner as in Example 8 with theexception of using the sialic acid derivative of Reference Example 16instead of the sialic acid derivative of Reference Example 14.

Example 13

A glycopeptide having a di-7-sialo derivative as 2,3-linked thereto,shown below, was obtained in the same manner as in Example 7 with theexception of using the asialooligosaccharide peptide not dansylated andobtained in Example 6.

Example 14

A glycopeptide having a di-7-sialo derivative as 2,6-linked thereto,shown below, was obtained in the same manner as in Example 8 with theexception of using the asialooligosaccharide peptide not dansylated andobtained in Example 6.

Example 15

A glycopeptide having a di-8-sialo derivative as 2,3-linked thereto,shown below, was obtained in the same manner as in Example 9 with theexception of using the asialooligosaccharide peptide not dansylated andobtained in Example 6.

Example 16

A glycopeptide having a di-8-sialo derivative as 2,6-linked thereto,shown below, was obtained in the same manner as in Example 10 with theexception of using the asialooligosaccharide peptide not dansylated andobtained in Example 6.

Example 17

A glycopeptide having a di-9-sialo derivative as 2,3-linked thereto,shown below, was obtained in the same manner as in Example 11 with theexception of using the asialooligosaccharide peptide not dansylated andobtained in Example 6.

Example 18

A glycopeptide having a di-9-sialo derivative as 2,6-linked thereto,shown below, was obtained in the same manner as in Example 12 with theexception of using the asialooligosaccharide peptide not dansylated andobtained in Example 6.

Example 19 Preparation ofHOOC-Ser-Thr-Thr-Asp-Asn(disialooligo)-Asp-Ile-Pro-NH₂

Asn(disialooligo) in the desired glycopeptide mentioned above means adisialooligoasparagine having sialic acid not protected with benzylgroup.

Into a solid-phase synthesis column was placed 50 mg of HMPA-PEGA resin,which was thoroughly washed with CH₂Cl₂ and DMF.

Fmoc-Ser(OtBu)-OH, 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT)and N-methylimidazole were dissolved in CH₂Cl₂, and the solution wasstirred for 5 minutes and thereafter placed into the solid-phasesynthesis column containing the resin, followed by stirring at roomtemperature for 3 hours. The resin was thereafter washed with methylenechloride, isopropanol and DMF and dried. The unreacted hydroxyl group onthe solid phase was thereafter acetylated using a 20% DMF solution ofacetic anhydride for 20 minutes for capping. The resin was washed withDMF and stirred along with a 20% piperidine/DMF solution for 20 minutesto remove the protective Fmoc group, whereby resin-Ser-NH₂ was obtained.The product was washed with DMF and dried.

Next, Fmoc-Thr(OtBu)-OH, Fmoc-Thr (OtBu)-OH and Fmoc-Asp(OtBu)-OH weresubjected to condensation in this order using HOBt.H₂O and PyBOP-DIPEA.After the condensation of the amino acids, the resulting reactionmixture was stirred along with a 20% piperidine/DMF (1:1) solution toremove the protective Fmoc group.

Subsequently, dibenzyl-Fmoc-asparagine-linked disialooligosaccharide wasdissolved in a 1:1 solvent mixture of DMSO and DMF, and the solution,HATU and DIPEA were stirred at room temperature for 24 hours forcondensation. The resulting resin was washed with DMF and thereafterstirred along with 10% acetic anhydride/2-propanol:methanol for 20minutes for capping. The resin was washed with DMF, a solvent mixture ofDMF and 2,6-lutidine (1:1) was then added to the resin, and TESOTf wasfurther added in an amount of 3 equiv. wt per oligosaccharide hydroxylgroup, followed by reaction for 1 hour to protect each oligosaccharidehydroxyl group with a TES (triethylsilyl) group.

The resin was washed with DMF and THF, and thereafter stirred along with20% piperidine/DMF for 20 minutes to remove the protective Fmoc group.The resin was washed with THF.

The resulting resin, and aspartic acid (Asp), isoleucine (Ile) andproline (Pro) were subjected to condensation in THF solvent usingHOBt.H₂O and PyBOP.DIPEA, and the protective Fmoc group was removed witha 20% piperidine/THF to obtain resin-Ser-Thr-Thr-Asp-Asn(TES-protecteddibenzyldisialooligo)-Asp-Ile-Pro-NH₂. Asn(TES-protecteddibenzyldisialooligo) mentioned means a disialooligoasparagine havingsialic acid with benzyl-protected carboxyl and having TES-protectedoligosaccharide hydroxyl group.

The resin resulting from condensation was thoroughly dried, andthereafter stirred along with a 95% aqueous solution of TFA at roomtemperature for 3 hours to cut off the protective group for aminoacid.TES and the resin. The resin was filtered off. The reaction mixturewas concentrated in a vacuum at room temperature, thereafter dissolvedin water and freeze-dried. The resulting product was dissolved in anaqueous solution of sodium hydroxide having a pH of 11 to hydrolyze thebenzyl ester for the removal of benzyl group, followed by neutralizationwith acetic acid. The product was freeze-dried as it was, and purifiedby HPLC to obtain the desired product, i.e.,HOOC-Ser-Thr-Thr-Asp-Asn(disialooligo)-Asp-Ile-Pro-NH₂.

(Mightsyl ODS-C18 250×20 mm, developing solvents A: 0.1% TFA aqueoussolution, B: 0.1% TFA acetonitrile:water=90:10, gradient A 100% O→B 100%60 min, flow rate 2.50 ml/min).

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to provide a processcapable of artificially and easily preparing a large amount of aglycopeptide having at least one asparagine-linked oligosaccharide ormucin-linked oligosaccharide at a desired position of the peptide chainthereof.

Further, according to the present invention, it is possible to obtain asialylglycopeptide which comprises an asparagine-linked oligosaccharidehaving sialic acid and wherein the sialic acid is not cut off from theglycopeptide by an acid treatment.

Further, according to the present invention, it is possible to obtainartificially and easily a large quantity of a glycopeptide having atleast one of various novel asparagine-linked oligosaccharides at adesired position of the peptide chain thereof, with sugar residuesremoved therefrom as desired.

Further, according to the present invention, it is possible to obtain aglycopeptide having sialic acid or a derivative thereof introduced intothe peptide with use of a sialic acid transferase.

1. A process for preparing a glycopeptide having at least oneasparagine-linked oligosaccharide at a desired position of the peptidechain thereof, the process comprising: (1) esterifying a hydroxyl groupof a resin having the hydroxyl group and a carboxyl group of an aminoacid having amino group nitrogen protected with a fat-soluble protectivegroup, (2) removing the fat-soluble protective group to form a freeamino group, (3) amidating the free amino group and a carboxyl group ofan amino acid having amino group nitrogen protected with a fat-solubleprotective group, (4) removing the fat-soluble protective group to forma free amino group, (5) repeating the steps (3) and (4) at least once,(6) amidating the free amino group and a carboxyl group of theasparagine portion of an asparagine-linked oligosaccharide having allthe hydroxyl groups unprotected and having amino group nitrogenprotected with a fat-soluble protective group, (7) removing thefat-soluble protective group to form a free amino group, (8) amidatingthe free amino group and a carboxyl group of an amino acid having aminogroup nitrogen protected with a fat-soluble protective group, (9)repeating the steps (7) and (8) at least once, (10) removing thefat-soluble protective group to form a free amino group, and (11)cutting off the resin with an acid.
 2. A process for preparing aglycopeptide having at least two asparagine-linked oligosaccharides at adesired position of the peptide chain thereof which comprises theprocess according to claim 1 wherein the steps (6) of amidating the freeamino group and a carboxyl group of the asparagine portion of anasparagine-linked oligosaccharide having all the hydroxyl groupsunprotected and having amino group nitrogen protected with a fat-solubleprotective group, and (7) of removing the fat-soluble protective groupto form a free amino group are additionally performed suitably.
 3. Aprocess for preparing a glycopeptide having at least oneasparagine-linked oligosaccharide at a desired position of the peptidechain thereof according to claim 1 wherein the steps (6) of amidatingthe free amino group and the carboxyl group of the asparagine portion ofan asparagine-linked oligosaccharide having all the hydroxyl groupsunprotected and having amino group nitrogen protected with a fat-solubleprotective group, and (7) of removing the fat-soluble protective groupto form a free amino group are performed as final steps.
 4. A processfor preparing a glycopeptide according to claim 1 wherein the step (1)of esterifying a hydroxyl group of a resin having the hydroxyl group anda carboxyl group of the asparagine portion of an asparagine-linkedoligosaccharide having all the hydroxyl groups unprotected and havingamino group nitrogen protected with a fat-soluble protective group isperformed in place of the step (6) or in addition to the step (6).
 5. Aprocess for preparing a glycopeptide according to claim 1 wherein theasparagine-linked oligosaccharide of the step (6) of claim 1 has atleast 6 sugar residues.
 6. A process for preparing a glycopeptideaccording to claim 1 wherein the asparagine-linked oligosaccharide ofthe step (6) of claim 1 has 9 to 11 sugar residues.
 7. A process forpreparing a glycopeptide according to claim 1 wherein theasparagine-linked oligosaccharide of the step (6) of claim 1 has atleast 6 sugar residues, and has a bifurcated oligosaccharide attachedthereto.
 8. A process for preparing a glycopeptide according to claim 1wherein the asparagine-linked oligosaccharide in (6) is anasparagine-linked asialooligosaccharide.
 9. A process for preparing aglycopeptide according to claim 1 wherein the fat-soluble protectivegroup is 9-fluorenylmethoxycarbonyl (Fmoc) group.
 10. A process forpreparing a glycopeptide according to claim 1 wherein a mucin-linkedoligosaccharide is used in place of a portion or the whole of theasparagine-linked oligosaccharide.
 11. A glycopeptide which isobtainable by a process according to claim 1 and which has at least oneasparagine-linked oligosaccharide or mutin-linked oligosaccharide at adesired position of the peptide chain thereof, and the glycopeptidehaving at least one oligosaccharide selected from amongasparagine-linked disialooligosaccharide and asparagine-linkedmonosialooligosaccharide attached as the asparagine-linkedoligosaccharide, and a carboxylic group of the sialic acid beingprotected with a protective group.
 12. A process for preparingglycopeptide having at least one asparagine-linked oligosaccharide at adesired position of the peptide chain thereof and a residue of sialicacid or a derivative thereof at a terminal end thereof, the processcomprising: (1) esterifying a hydroxyl group of a resin having thehydroxyl group and a carboxyl group of an amino acid having amino groupnitrogen protected with a fat-soluble protective group, (2) removing thefat-soluble protective group to form a free amino group, (3) amidatingthe free amino group and a carboxyl group of an amino acid having aminogroup nitrogen protected with a fat-soluble protective group, (4)removing the fat-soluble protective group to form a free amino group,(5) repeating the steps (3) and (4) at least once, (6) amidating thefree amino group and a carboxyl group of the asparagine portion of anasparagine-linked oligosaccharide having all the hydroxyl groupsunprotected and having amino group nitrogen protected with a fat-solubleprotective group, (7) removing the fat-soluble protective group to forma free amino group, (8) amidating the free amino group and a carboxylgroup of an amino acid having amino group nitrogen protected with afat-soluble protective group, (9) repeating the steps (7) and (8) atleast once, (10) removing the fat-soluble protective group to form afree amino group, (11) cutting off the resin with an acid, and (12)transferring sialic acid or a derivative thereof to the resultingglycopeptide using a sialic acid transferase.
 13. A process forpreparing a glycopeptide according to claim 12 wherein a marker isreacted with the resin before the resin is cut off with the acid in step(11).
 14. A process for preparing a glycopeptide according to claim 13wherein the marker is a dansyl halide.
 15. A process for preparing5-acetamido-3,5,7-trideoxy-7-fluoro-D-glycero-β-D-lacto-2-nonulopyranosidonicacid comprising reacting N-acetyl-4-deoxy-4-fluoro-D-mannosamine, sodiumpiruvate, bovine serum albumin and aldolase sialate.
 16. A process forpreparing5-acetamido-3,5,7-trideoxy-7-fluoro-D-glycero-β-D-lacto-2-nonulopyranosidonicacid comprising hydrogenating benzyl2-azido-2,4-dideoxy-4-fluoro-β-D-mannopyranoside in the presence ofacetic anhydride to obtain N-acetyl-4-deoxy-4-fluoro-D-mannosamine, andsubsequently reacting the product with sodium piruvate, bovine serumalbumin and aldolase sialate.